K-Ras Promotes Tumorigenicity through Suppression of Non-canonical Wnt Signaling.

K-Ras and H-Ras share identical effectors and have similar properties; however, the high degree of tumor-type specificity associated with K-Ras and H-Ras mutations suggests that they have unique roles in oncogenesis. Here, we report that oncogenic K-Ras, but not H-Ras, suppresses non-canonical Wnt/Ca(2+) signaling, an effect that contributes strongly to its tumorigenic properties. K-Ras does this by binding to calmodulin and so reducing CaMKii activity and expression of Fzd8. Restoring Fzd8 in K-Ras mutant pancreatic cells suppresses malignancy, whereas depletion of Fzd8 in H-Ras(V12)-transformed cells enhances their tumor initiating capacity. Interrupting K-Ras-calmodulin binding using genetic means or by treatment with an orally active protein kinase C (PKC)-activator, prostratin, represses tumorigenesis in K-Ras mutant pancreatic cancer cells. These findings provide an alternative way to selectively target this "undruggable" protein.

The Diagnostic Value of microRNA Expression Analysis in Detecting Intraductal Papillomas in Patients with Pathological Nipple Discharge.

Patients with pathological nipple discharge (PND) often undergo local surgical procedures because standard radiologic imaging fails to identify the underlying cause. MicroRNA (MiRNA) expression analysis of nipple fluid holds potential for distinguishing between breast diseases. This study aimed to compare miRNA expression levels between nipple fluids from patients with PND to identify possible relevant miRNAs that could differentiate between intraductal papillomas and no abnormalities in the breast tissue. Nipple fluid samples from patients with PND without radiological and pathological suspicion for malignancy who underwent a ductoscopy procedure were analyzed. We used univariate and multivariate regression analyses to identify nipple fluid miRNAs differing between pathologically confirmed papillomas and breast tissue without abnormalities. A total of 27 nipple fluid samples from patients with PND were included for miRNA expression analysis. Out of the 22 miRNAs examined, only miR-145-5p was significantly differentially expressed (upregulated) in nipple fluid from patients with an intraductal papilloma compared to patients showing no breast abnormalities (OR 4.76, p = 0.046), with a diagnostic accuracy of 92%. miR-145-5p expression in nipple fluid differs for intraductal papillomas and breast tissue without abnormalities and, therefore, has potential as a diagnostic marker to signal presence of papillomas in PND patients. However, further refinement and validation in clinical trials are necessary to establish its clinical applicability.

Immune delineation of laryngeal papilloma reveals enhanced neutrophil associated gene profile.

Laryngeal papilloma (LP) is a rare benign disease, caused by recurrent multisite papillomas that are referred to as recurrent respiratory papillomatosis (RRP). RRP is caused primarily by two types of human papillomavirus (HPV): HPV6 and HPV11. The immune dysregulation within the microenvironment of the lesions has been shown to likely play a role in the development of RRP. The present study aimed at analyzing the transcriptional profile of immune response genes and cancer-related genes in the LP microenvironment. We used the NanoString® nCounter® analysis system to study expression of 730 genes among seven paired samples of LP and healthy laryngeal (HL) tissue. qRT-PCR and flow cytometric analysis was performed to confirm identified transcripts and follow-up scores of infiltrating immune cells, respectively. In total, 113 differentially expressed transcripts were detected of which 37 showed increased expression levels and 76 decreased expression levels in the LP samples compared to the HL samples (fold change ≥ 2). Transcripts with increased expression levels included S100As (A7, A8, and A12), CEACAM1, neutrophil activation associated cytokines (IL8), chemokines (CXCL6), and IL receptors, e.g., IL4R. Transcripts with decreased expression in LP were associated with innate and adaptive immunity. Overall, HPV6 and 11 were present in 67% and 33% of the patients, respectively. There was a significant increase in neutrophils and a significant decrease in CD8+ T cells in LP. LP samples display an immune profile characterized by enhanced expression of neutrophilic markers and significantly reduced T cell-associated markers.

Epidermal ROCK2 induces AKT1/GSK3ß/ß-catenin, NFκB and dermal tenascin C; but enhanced differentiation and p53/p21 inhibit papilloma.

ROCK2 roles in epidermal differentiation and carcinogenesis have been investigated in mice expressing an RU486-inducible, 4HT-activated ROCK2 transgene (K14.creP/lslROCKer). RU486/4HT-mediated ROCKer activation induced epidermal hyperplasia similar to cutaneous oncogenic rasHa (HK1.ras); however ROCKer did not elicit papillomas. Instead, anomalous basal-layer ROCKer expression corrupted normal ROCK2 roles underlying epidermal rigidity/stiffness and barrier maintanance, resulting in premature keratin K1, loricrin and filaggrin expression. Also, hyperproliferative/stress-associated keratin K6 was reduced; possibly reflecting altered ROCK2 roles in epidermal rigidity and keratinocyte flexibility/migration during wound healing. Consistent with increased proliferation, K14.creP/lslROCKer hyperplasia displayed supra-basal-to-basal increases in activated p-AKT1, inactivated p-GSK3ß ser9 and membranous/nuclear ß-catenin expression together with weak NFκB, which were absent in equivalent HK1.ras hyperplasia. Furthermore, ROCKer-mediated increases in epidermal rigidity via p-MypT1 inactivation/elevated MLC, coupled to anomalous ß-catenin expression, induced tenascin C-positive dermal fibroblasts. Alongside an altered ECM, these latent tenascin C-positive dermal fibroblasts may become putative pre-cancer-associated fibroblasts (pre-CAFs) and establish a susceptibility that subsequently contributes to tumour progression. However, anomalous differentiation was also accompanied by an immediate increase in basal-layer p53/p21 expression; suggesting that while ROCK2/AKT1/ß-catenin activation increased keratinocyte proliferation resulting in hyperplasia, compensatory p53/p21 and accelerated differentiation helped inhibit papillomatogenesis.

Relationship between inflammation and the severity of Recurrent Respiratory Papillomatosis.

OBJECTIVE: To characterize inflammatory cells in Recurrent Respiratory Papillomatosis (RRP) and to correlate it with severity using the Derkay laryngoscopic scale. MATERIALS AND METHODS: The data and biopsies from 36 patients with Juvenile (JRRP) and 56 patients with Adult (ARRP) were collected and analyzed under light microscopy. The patients were separated into groups according to the Derkay index: ≥20 for the most severe and < 20 for the less severe cases. Immunohistochemical analysis using CD3, CD4, CD8, CD15, CD20, CD68, FoxP3 and MUM-1 antibodies was performed, and the inflammatory cells were quantified. All the clinicopathological characteristics and the results of the immunohistochemical analysis were compared among the groups proposed using the Chi-Square test and correlated through the Spearman correlation test. RESULTS: The ARRP showed significantly higher quantities of CD3+, CD8+ and MUM1+ cells (p < .05) than the JRRP samples. The presence of CD15+ cells showed positive correlation with the Derkay index (p < .05), while the MUM-1+ cells showed an inverse correlation (p = .01). CONCLUSION: There are differences between the inflammatory cells population in the juvenile and adult groups and it can be related to disease severity.

Association of Epithelial Atypia With Recurrence After Surgical Excision in Conjunctival Papilloma.

PURPOSE: To investigate the association between recurrence of conjunctival papillomas and presence of atypical epithelial changes in patients undergoing surgical excision for conjunctival papilloma. METHODS: We retrospectively reviewed 1,195 ophthalmic pathology specimens from 2004 to 2014 at Ichikawa General Hospital. Pathologic specimens of 5 patients with a final diagnosis of "conjunctival papilloma" were stained with hematoxylin-eosin, Ki 67, p53, human papillomavirus (HPV) 16 and 18 antibodies. RESULTS: Of 1,195 patients, 5 patients (4 men, 1 woman; age range: 27∼57 years, mean age: 38.4 years) had a diagnosis of conjunctival papilloma, which constituted to 0.42% of the pathologic diagnosis made for the ophthalmology specimens. All specimens displayed multiple fronds of thickened conjunctival epithelium that enclosed cores of vascularized connective tissues. Three patients with recurrence after surgical excision demonstrated moderate to severe epithelial atypia, who also showed higher staining with Ki67 and p53 compared with patients with no recurrence. HPV16 and 18 antibodies staining did not appear to relate to recurrences. CONCLUSIONS: Conjunctival papillomas with higher positive staining for Ki67 and p53 seem to have a higher risk of recurrence even after complete surgical excision and necessitate careful follow-up.

Poly-MVA attenuates 7,12- dimethylbenz[a]anthracene initiated and croton oil promoted skin papilloma formation on mice skin.

OBJECTIVE: Chemopreventive agents which exhibit activities such as anti-inflammation, inhibition of carcinogen induced mutagenesis and scavenging of free radical might play a decisive role in the inhibition of chemical carcinogenesis either at the initiation or promotion stage. Many synthesized palladium (Pd) complexes tested experimentally for antitumor activity are found effective. Poly-MVA is a liquid blend preparation containing B complex vitamins, ruthenium with Pd complexed with alpha lipoic acid as the major ingredients. The antitumor effect of Poly-MVA was evaluated against 7,12-dimethylbenz[a] anthracene-initiated croton oil-promoted papilloma formation on mice skin. Skin tumor was initiated with a single application of 390 nmol of DMBA in 20 µl acetone. The effect of Poly-MVA against croton oil- induced inflammation and lipid peroxidation on the mice skin was also evaluated. Topical application of Poly-MVA (100 µl, twice weekly for 18 weeks) 30 minutes prior to each croton oil application, significantly decreased the tumor incidence (11%) and the average number of tumor per animals. Application of Poly-MVA (100 µl) before croton oil significantly (p &#60; 0.05) protected the mouse skin from inflammation (36%) and lipid peroxidation (14%) when compared to the croton oil alone treated group. Experimental results indicate that Poly-MVA attenuate the tumor promoting effects of croton oil and the effect may probably be due to its anti-inflammatory and antioxidant activity.

Expression of p16Ink4a in mixed squamous cell and glandular papilloma of the lung.

Mixed squamous cell and glandular papilloma (mixed papilloma) of the lung is an extremely rare neoplasm, with only 21 cases reported in the English literature. Although the expression of p16Ink4a has been analyzed in only two cases of mixed papilloma, they were negative for p16Ink4a . Therefore, the significance of p16Ink4a overexpression in mixed papilloma remains unclear. This is the first case of mixed papilloma with positive p16Ink4a expression in a 72-year-old male smoker. The 20 mm sized tumor was histologically diagnosed as mixed papilloma following right upper lobectomy. Immunohistochemically, cytokeratin 5 and p40 positivity was predominant in basal cells of the glandular component and squamous cells, while thyroid transcription factor-1, p53, and Ki-67 were focally positive. Both glandular and squamous components were diffusely positive for p16Ink4a . This finding could be important to clarify the pathogenesis and biology of mixed papilloma.

Infiltrating epitheliosis of the breast: characterization of histological features, immunophenotype and genomic profile.

AIMS: Infiltrating epitheliosis is a rare complex sclerosing lesion (CSL) of the breast, characterized by infiltrating ducts immersed in a scleroelastotic stroma and filled with cells having architectural and cytological patterns reminiscent of those of usual ductal hyperplasia. In this study we sought to define the molecular characteristics of infiltrating epitheliosis. METHODS AND RESULTS: Eight infiltrating epitheliosis, adjacent breast lesions (one usual ductal hyperplasia, one papilloma, one micropapillary ductal carcinoma in situ and one low-grade adenosquamous carcinoma), and corresponding normal breast tissue from each case were microdissected and subjected to massively parallel sequencing analysis targeting all coding regions of 254 genes mutated recurrently in breast cancer and/or related to DNA repair. Mutations in components of the PI3K pathway were found in all infiltrating epitheliosis samples, seven of which harboured PIK3CA hotspot mutations, while the remaining case displayed a PIK3R1 somatic mutation. CONCLUSIONS: Somatic mutations affecting PI3K pathway genes were found to be highly prevalent in infiltrating epitheliosis, suggesting that these lesions may be neoplastic rather than hyperplastic. The landscape of somatic genetic alterations found in infiltrating epitheliosis is similar to that of radial scars/CSLs, suggesting that infiltrating epitheliosis may represent one end of this spectrum of lesions.

Immunohistochemical Expression of Cyclin D1, Cytokeratin 20, and Uroplakin III in Proliferative Urinary Bladder Lesions Induced by o-Nitroanisole in Fischer 344/N Rats.

o-Nitroanisole is an intermediate in the manufacture of azo dyes. In a National Toxicology Program stop-exposure study,o-nitroanisole induced hyperplasia, papillomas, and papillary carcinomas in the urinary bladder of Fischer 344/N rats.o-Nitroanisole was investigated since occupational or environmental exposure to aniline and azo dyes is a risk factor for urinary bladder cancer in humans. The current study describes the morphology of urinary bladder neoplasms seen in rats with respect to those observed in humans. This study also evaluated immunohistochemical expression of the cell cycle-related proteins cyclin D1 and p53 and the differentiation markers cytokeratin 20 and uroplakin III in hyperplastic (n= 11) and neoplastic (n= 6 papillomas,n= 11 carcinomas) lesions of the urinary bladder epithelium from rats treated with o-nitroanisole and in normal (n= 6) urinary bladders from untreated rats. The tumors observed were more similar to the papillary type rather than the muscle-invasive type of urinary bladder cancer in humans. The preneoplastic and neoplastic lesions observed suggest progression from hyperplasia to papilloma to papillary carcinoma. With neoplastic progression (hyperplasia to papilloma to carcinoma), cyclin D1 immunoreactivity progressively increased in intensity, percentage of cells staining, and distribution. Overexpression of p53 was not found. Cytokeratin 20 staining decreased in superficial cells, while uroplakin III staining increased in intermediate and basal cells with progression from hyperplasia to carcinoma. The results are consistent with increased cell cycle dysregulation or proliferation (cyclin D1), decreased differentiation (cytokeratin 20), and abnormal differentiation (uroplakin III) as lesions progress toward malignancy.

Attenuation of DMBA/croton oil induced mouse skin papilloma by Apodytes dimidiata mediated by its antioxidant and antimutagenic potential.

Context Considering the role of cellular oxidative stress in mutations and subsequent transformation, phytochemicals with antioxidant potential has become a primary choice as chemopreventives. Apodytes dimidiata E. Mey. Ex. Arn (Icacinaceae), a widely used plant in Zulu traditional medicine, is reported to possess antioxidant activity. Objective To investigate the chemopreventive efficacy of methanol extract of A. dimidiata leaf (AMF). Materials and methods Antimutagenic potential of AMF (25, 50 and 75 µg/plate) was evaluated by the Ames test. The ability of AMF (100 and 250 mg/kg orally) on restoration of depleted antioxidant status by sodium fluoride (NaF) was analysed on BALB/c mice. 7,12-Dimethylbenz[a]anthracene/croton oil induced mouse skin papilloma model was studied up to 20 weeks to analyse the anticarcinogenic effect of AMF (1%, 3% and 5% topically, twice weekly for 6 weeks). Phytochemicals of AMF were characterized by GC-MS. Results AMF (75 µg/plate) reverted 4-nitro-o-phenylenediamine (NPDA) induced mutations in Salmonella typhimurium strains, TA 98, 100 and 102 by 74.8%, 72.5% and 69.3%, respectively. Against sodium azide, the percentage reversion was 80.4, 71.3 and 71.3. In mice, AMF (250 mg/kg for 4 days) increased the serum superoxide dismutase (SOD) and catalase activities by 48.71% and 30.3% against the NaF-induced drop. GSH level was improved by 48.59% with a concomitant decrease in TBARS (57.67%). The skin papilloma reduction was 79.32% for 5% AMF. Squalene, dodecanoic, tetradecanoic and hexadecanoic acids are the known antioxidant and chemopreventive molecules identified by GC-MS. Discussion and conclusion Antioxidant and antimutagenic activities of AMF might have contributed to its anticarcinogenic potential.

Altered expression of CKs 14/20 is an early event in a rat model of multistep bladder carcinogenesis.

Cytokeratins (CKs) 14 and 20 are promising markers for diagnosing urothelial lesions and for studying their prognosis and histogenesis. This work aimed to study the immunohistochemical staining patterns of CK14/20 during multistep carcinogenesis leading to papillary bladder cancer in a rat model. Thirty female Fischer 344 rats were divided into three groups: group 1 (control); group 2, which received N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) for 20 weeks plus 1 week without treatment; and group 3, which received BBN for 20 weeks plus 8 weeks without treatment. Bladder lesions were classified histologically. CK14 and CK20 immunostaining was assessed according to its distribution and intensity. In control animals, 0-25% of basal cells and umbrella cells stained positive for CK14 and CK20 respectively. On groups 2 and 3, nodular hyperplastic lesions showed normal CK20 and moderately increased CK14 staining (26-50% of cells). Dysplasia, squamous metaplasia, papilloma, papillary tumours of low malignant potential and low- and high-grade papillary carcinomas showed increased CK14 and CK20 immunostaining in all epithelial layers. Altered CK14 and CK20 expression is an early event in urothelial carcinogenesis and is present in a wide spectrum of urothelial superficial neoplastic and preneoplastic lesions.

Identification of microRNAs in Nipple Discharge as Potential Diagnostic Biomarkers for Breast Cancer.

BACKGROUND: Intraductal breast cancer is generally difficult to diagnose because of a lack of an efficient method for detection. The purpose of this study was to reveal and validate the differential expression of microRNAs (miRNAs) in nipple discharge from intraductal papilloma patients and identify miRNAs as novel potential biomarkers for primary breast cancer. METHODS: Nipple discharge samples were collected from three intraductal carcinoma breast cancer patients and three intraductal papilloma patients. The initial screening of miRNA expression was performed with an Axon GenePix 4000B microarray scanner using a novel approach to label miRNAs. The expression levels of the miRNAs selected from the initial screening were further examined by quantitative real-time polymerase chain reaction (qRT-PCR) in 21 validation samples (8 carcinomas and 13 benign tumors). An independent t test was used to detect significant correlations between the miRNA expression levels and breast cancer. RESULTS: Microarray profiling demonstrated that three miRNAs were markedly up-regulated and three miRNAs were down-regulated in the intraductal carcinoma breast cancer patients compared to the papilloma group. The qRT-PCR analysis further verified that four miRNAs (miR-4484, miR-K12-5-5p, miR-3646, and miR-4732-5p) might serve as potential tumor biomarkers for breast cancer detection. CONCLUSION: The novel approach of using a microarray scanner is applicable for studying biomarkers in nipple discharge containing small amounts of miRNA. miRNAs could serve as potential tumor biomarkers that can assist in breast cancer screening. Up-regulation of miR-4484, miR-K12-5-5p, and miR-3646 in nipple discharge may be a predictor of malignant breast cancer.

Activation-induced cytidine deaminase is dispensable for virus-mediated liver and skin tumor development in mouse models.

Activation-induced cytidine deaminase (AID) not only promotes immune diversity by initiating somatic hypermutation and class switch recombination in immunoglobulin genes but also provokes genomic instability by introducing translocations and mutations into non-immunoglobulin genes. To test whether AID is essential for virus-induced tumor development, we used two transgenic tumor models: mice expressing hepatitis C virus (HCV) core proteins (HCV-Tg), driven by the hepatitis B virus promoter, and mice expressing human papillomavirus type 8 proteins (HPV8-Tg), driven by the Keratin 14 promoter. Both strains were analyzed in the absence and presence of AID by crossing each with AID (-/-) mice. There was no difference in the liver tumor frequency between the HCV-Tg/AID (+/+) and HCV-Tg/AID (-/-) mice at 20 months of age although the AID (+/+) mice showed more severe histological findings and increased cytokine expression. Furthermore, a low level of AID transcript was detected in the HCV-Tg/AID (+/+) liver tissue that was not derived from hepatocytes themselves but from intra-hepatic immune cells. Although AID may not be the direct cause of HCV-induced oncogenesis, AID expressed in B cells, not in hepatocytes, may prolong steatosis and cause increased lymphocyte infiltration into HCV core protein-induced liver lesions. Similarly, there was no difference in the time course of skin tumor development between the HPV8-Tg/AID (-/-) and HPV8-Tg/AID (+/+) groups. In conclusion, AID does not appear to be required for tumor development in the two virus-induced tumor mouse models tested although AID expressed in infiltrating B cells may promote inflammatory reactions in HCV core protein-induced liver pathogenesis.

Activation of MEK2 is sufficient to induce skin papilloma formation in transgenic zebrafish.

BACKGROUND: Mutations in mitogen-activated protein kinase (MAPK) kinase 1 (MEK1) that occur during cell proliferation and tumor formation are well described. Information on the roles of MEK2 in these effects is still limited. We established a constitutive MEK2 transgenic zebrafish, Tg(krt14:MEK2S219D-GFP), to elucidate the role of MEK2 in skin tumor formation. RESULTS: We found that both constitutive MEK2 and MEK1 are able to phosphorylate the extracellular signal-regulated kinase 1 (ERK1) protein. Transient expression of constitutive MEK2 and MEK1 in the zebrafish epidermis induced papillary formation at 48 h post-fertilization, but no effects were observed due to the expression of MEK1, MEK2, or the dominant negative form of MEK2. The transgenic zebrafish, Tg(krt14:MEK2S219D-GFP), developed skin papillomas in the epidermis within 6 days post-fertilization (dpf). The phospho-ERK signal was detected in section of skin papillomas in an immunohistochemical experiment. Treatment with 50 µM of the MEK inhibitor, U0126, had significantly decreased the skin papilloma formation in Tg(krt14:MEK2S219D-GFP) zebrafish by 6 dpf. In vitro and in vivo proliferation assay in COS-1 cells and in Tg(krt14:MEK2S219D-GFP) transgenic fish show significantly increased cell number and Ki-67 signaling. CONCLUSION: Our data indicate that MEK2 is sufficient to induce epidermal papilloma formation through MAPK signaling in zebrafish, and this transgenic model can be used as a new platform for drug screening.

Keeping order in the neighborhood: new roles for TGFbeta in maintaining epithelial homeostasis.

TGFbetas are thought to have tumor suppressor activity in many organ systems, but receptor inactivation in mouse models has not previously resulted in increased spontaneous tumorigenesis. A study in this issue of Cancer Cell shows that mice with a targeted knockout of the type II TGFbeta receptor in stratified epithelia specifically develop spontaneous squamous cell carcinomas in the anogenital region, but not in the skin. Loss of TGFbeta signaling appears to destabilize the epithelium such that homeostasis fails in the face of persistent proliferative challenge, a normal feature of the anogenital site, and latent invasive and migratory phenotypes are unmasked.

Loss of TGFbeta signaling destabilizes homeostasis and promotes squamous cell carcinomas in stratified epithelia.

Although TGFbeta is a potent inhibitor of proliferation, epithelia lacking the essential receptor (TbetaRII) for TGFbeta signaling display normal tissue homeostasis. By studying asymptomatic TbetaRII-deficient stratified epithelia, we show that tissue homeostasis is maintained by balancing hyperproliferation with elevated apoptosis. Moreover, rectal and genital epithelia, which are naturally proliferative, develop spontaneous squamous cell carcinomas with age when TbetaRII is absent. This progression is associated with a reduction in apoptosis and can be accelerated in phenotypically normal epidermis by oncogenic mutations in Ras. We show that TbetaRII deficiency leads to enhanced keratinocyte motility and integrin-FAK-Src signaling. Together, these mechanisms provide a molecular framework to account for many of the characteristics of TbetaRII-deficient invasive SQCCs.

Apoptotic effect of pheophorbide a-mediated photodynamic therapy on DMBA/TPA-induced mouse papillomas.

Pheophorbide a (Pa) is a chlorine-based photosensitizer, and Pa-mediated photodynamic therapy (PDT) reportedly exhibits antitumor activity against various malignancies. The aim of our study was to investigate the therapeutic effect of Pa-mediated PDT on 7,12-dimethylbenz[a]anthracene (DMBA)/12-O-tetradecanoylphorobol-13-acetate (TPA)-induced mouse papillomas. Thirty mice received a topical application of DMBA/TPA on their backs to induce mouse papillomas. One week after two sessions of Pa-mediated PDT, immunohistochemical stains and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay were performed to evaluate the apoptotic effects thereof on the papillomas. Among 63 mouse papillomas treated with Pa-mediated PDT, 17.5% of the lesions were completely removed 1 week after the first treatment, while 31.7% disappeared 1 week after the second treatment. Statistical analyses revealed significant differences in therapeutic outcomes for the Pa-mediated PDT group in comparison to a solvent-PDT group and a Pa group. Additionally, a marked downregulation of proliferating cell nuclear antigen expression, as well as upregulation of cleaved caspase 3 and cleaved poly(ADP-ribose) polymerase expression, was noted in the Pa-PDT group, compared to the solvent-PDT group and Pa group. TUNEL assay revealed higher apoptotic cell counts in the Pa-PDT group, although the difference was not statistically significant. Our data demonstrated that Pa-mediated PDT is effective in treating DMBA/TPA-induced mouse papillomas.

P16(INK4A) immunostaining is a strong indicator for high-risk-HPV-associated oropharyngeal carcinomas and dysplasias, but is unreliable to predict low-risk-HPV-infection in head and neck papillomas and laryngeal dysplasias.

Human papillomavirus (HPV) is a risk factor for the development of benign and malignant mucosal head and neck lesions. P16(INK4A) is often used as a surrogate marker for HPV-infection, although there is still controversy with respect its reliability. Our aim was to determine if p16(INK4A) overexpression can accurately predict both high-risk and low-risk-HPV-presence in (pre)malignant and benign head and neck lesions. P16(INK4A) immunohistochemistry was performed on paraffin-embedded tissue sections of 162 oropharyngeal squamous cell carcinomas (OPSCC), 14 tonsillar and 23 laryngeal dysplasias, and 20 tonsillar and 27 laryngeal papillomas. PCR, enzyme-immunoassay and FISH analysis were used to assess HPV-presence and type. Of the 162 OPSCC and 14 tonsillar dysplasias, 51 (31%) and 10 (71%) were HPV16-positive, respectively. All tonsillar papillomas were HPV-negative and four laryngeal dysplasias and 26 laryngeal papillomas were positive for HPV6 or -11. P16(INK4A) immunohistochemistry revealed a strong nuclear and cytoplasmic staining in 50 out of 51 HPV16-positive and 5 out of 111 HPV-negative OPSCC (p < 0.0001) and in all HPV16-positive tonsillar dysplasias, whereas highly variable staining patterns were detected in the papillomas and laryngeal dysplasias, irrespective of the HPV-status. In addition, the latter lesions generally showed a higher nuclear than cytoplasmic p16(INK4A) immunostaining intensity. In conclusion, our data show that strong nuclear and cytoplasmic p16(INK4A) overexpression is a reliable surrogate indicator for HPV16 in OPSCC and (adjacent) dysplasias. For HPV6 or -11-positive and HPV-negative benign and premalignant lesions of the tonsil and larynx, however, p16(INK4A) immunostaining is highly variable and cannot be recommended to predict HPV-presence.

Skp2 deficiency inhibits chemical skin tumorigenesis independent of p27(Kip1) accumulation.

S-phase kinase-associated protein 2 (Skp2) functions as the receptor component of the Skp-Cullin-F-box complex and is implicated in the degradation of several cell cycle regulators, such as p21(Cip1), p27(Kip1), p57(Kip2), and cyclin E. Numerous studies in human and experimental tumors have demonstrated low p27(Kip1) levels and elevated Skp2 expression. However, a direct association between the inverse correlation of Skp2 and p27(Kip1) with tumorigenesis has not been demonstrated. Herein, we provide evidence that skin tumorigenesis is inhibited in Skp2(-/-) mice. An analysis of mouse keratinocytes indicates that increased p27(Kip1) levels in Skp2(-/-) epidermis cause reduced cell proliferation that is alleviated in the epidermis from Skp2(-/-)/p27(-/-) compound mice. In contrast, we establish that a p27(Kip1) deficiency does not overturn the reduced skin tumorigenesis experienced by Skp2(-/-) mice. In addition, Skp2(-/-) epidermis exhibits an accumulation of p53-cofactor CBP/p300 that is associated with elevated apoptosis in hair follicles and decreased skin tumorigenesis. We conclude that p27(Kip1) accumulation is responsible for the hypoplasia observed in normal tissues of Skp2(-/-) mice but does not have a preponderant function in reducing skin tumorigenesis.