Activating FGFR3 mutations cause mild hyperplasia in human skin,but are insufficient to drive benign or malignant skin tumors

Fibroblast growth factor receptor 3 (FGFR3) activating mutations are drivers of malignancy in several human tissues, including bladder, lung, cervix, and blood. However, in skin, these mutations are associated predominantly with benign, common epidermal growths called seborrheic keratoses (SKs). How epidermis resists FGFR3 mediated transformation is unclear, but previous studies have suggested that FGFR3 activation in skin keratinocytes may serve a tumor-suppressive role by driving differentiation and antagonizing Ras signaling. To define the role of FGFR3 in human normal and neoplastic epidermis, and to directly test the hypothesis that FGFR3 antagonizes Ras, we engineered human skin grafts in vivo with mutant active FGFR3 or shRNA FGFR3 knockdown. We show that FGFR3 active mutants drive mild hyperproliferation, but are insufficient to support benign or malignant tumorigenesis, either alone, or in combination with G₁–S checkpoint release. This suggests that additional cell-intrinsic or stromal cues are required for formation of benign SKs with FGFR3 mutations. Further, FGFR3 activation does not alter the growth kinetics or differentiation status of engineered human epidermal SCCs driven by Ras, and FGFR3 protein itself is dispensable for Ras-driven SCC. To extend these findings to patients, we examined a uniquely informative human tumor in which SCC developed in continuity with a SK, raising the hypothesis that one of the tumors evolved from the other. However, mutational analysis from each tumor indicates that the overlapping SK and SCC evolved independently and supports our conclusion that FGFR3 activation is insufficient to drive SCC.     

A miR‐34a‐SIRT6 axis in the squamous cell differentiation network

Squamous cell carcinomas (SCCs) are highly heterogeneous tumours, resulting from deranged expression of genes involved in squamous cell differentiation. Here we report that microRNA‐34a (miR‐34a) functions as a novel node in the squamous cell differentiation network, with SIRT6 as a critical target. miR‐34a expression increases with keratinocyte differentiation, while it is suppressed in skin and oral SCCs, SCC cell lines, and aberrantly differentiating primary human keratinocytes (HKCs). Expression of this miRNA is restored in SCC cells, in parallel with differentiation, by reversion of genomic DNA methylation or wild‐type p53 expression. In normal HKCs, the pro‐differentiation effects of increased p53 activity or UVB exposure are miR‐34a‐dependent, and increased miR‐34a levels are sufficient to induce differentiation of these cells both in vitro and in vivo. SIRT6, a sirtuin family member not previously connected with miR‐34a function, is a direct target of this miRNA in HKCs, and SIRT6 down‐modulation is sufficient to reproduce the miR‐34a pro‐differentiation effects. The findings are of likely biological significance, as SIRT6 is oppositely expressed to miR‐34a in normal keratinocytes and keratinocyte‐derived tumours.     

Aberrant Expression of Osteopontin and E-Cadherin Indicates Radiation Resistance and Poor Prognosis for Patients with Cervical Carcinoma

Radiotherapy is the first-line treatment for all stages of cervical cancer, whether it is used for radical or palliative therapy. However, radioresistance of cervical cancer remains a major therapeutic problem. Consequently, we explored if E-cadherin (a marker of epithelial-mesenchymal transition) and osteopontin could predict radioresistance in patients with locally advanced cervical squamous cell carcinoma (LACSCC). Patients were retrospectively reviewed and 111 patients divided into two groups (radiation-resistant and radiation-sensitive groups) according to progression-free survival (PFS). In pretreated paraffin-embedded tissues, we evaluated E-cadherin and osteopontin expression using immunohistochemical staining. The percentage of patients with high osteopontin but low E-cadherin expression in the radiation-resistant group was significantly higher than those in the radiation-sensitive group (p<0.001). These patients also had a lower 5-year PFS rate (p<0.001). Our research suggests that high osteopontin but low E-cadherin expression can be considered as a negative, independent prognostic factor in patients with LACSCC ([Hazard ratios (95% CI) 6.766 (2.940, 15.572)], p<0.001).     

Pannexin1 and Pannexin3 Exhibit Distinct Localization Patterns in Human Skin Appendages and are Regulated during Keratinocyte Differentiation and Carcinogenesis

Having shown that Panx1 and Panx3 are expressed in the epidermis, we investigated their distribution in human skin adnexal structures and skin cancer. Both proteins were found in hair follicles, sebaceous and eccrine glands, as well as blood vessels. Panx1 was detected as punctate or diffuse intracellular labeling, while Panx3 was only observed as diffuse intracellular staining, suggesting different functions. We also identified the Panx3 immunoreactive ～70 kD species modulated during keratinocyte differentiation as Panx3. Since our data indicate that pannexins are regulated during keratinocyte differentiation, we assessed whether their levels are altered under circumstances in which keratinocyte differentiation is compromised. We found that Panx1 and Panx3 levels are highly reduced in human keratinocyte tumors, thus showing for the first time that both pannexins are dysregulated in human cancers. Altogether, these data suggest that Panx1 and Panx3 have distinct and unique functions within the skin in health and disease.     

Pregnancy-specific glycoprotein expression in normal gastrointestinal tract and in tumors detected with novel monoclonal antibodies

Pregnancy-specific glycoproteins (PSGs) are immunoglobulin superfamily members related to the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family and are encoded by 10 genes in the human. They are secreted at high levels by placental syncytiotrophoblast into maternal blood during pregnancy, and are implicated in immunoregulation, thromboregulation, and angiogenesis. To determine whether PSGs are expressed in tumors, we characterized 16 novel monoclonal antibodies to human PSG1 and used 2 that do not cross-react with CEACAMs to study PSG expression in tumors and in the gastrointestinal (GI) tract using tissue arrays and immunohistochemistry. Staining was frequently observed in primary squamous cell carcinomas and colonic adenocarcinomas and was correlated with the degree of tumor differentiation, being largely absent from metastatic samples. Staining was also observed in normal oesophageal and colonic epithelium. PSG expression in the human and mouse GI tract was confirmed using quantitative RT-PCR. However, mRNA expression was several orders of magnitude lower in the GI tract compared to placenta. Our results identify a non-placental site of PSG expression in the gut and associated tumors, with implications for determining whether PSGs have a role in tumor progression, and utility as tumor biomarkers.     

Lysyl oxidase-like 2 represses Notch1 expression in the skin to promote squamous cell carcinoma progression

Lysyl oxidase-like 2 (LOXL2) is involved in a wide range of physiological and pathological processes, including fibrosis and tumor progression, implicating intracellular and extracellular functions. To explore the specific in vivo role of LOXL2 in physiological and tumor contexts, we generated conditional gain- and loss-of-function mouse models. Germ-line deletion of Loxl2 promotes lethality in half of newborn mice mainly associated to congenital heart defects, while Loxl2 overexpression triggers male sterility due to epididymal dysfunction caused by epithelial disorganization, fibrosis and acute inflammation. Remarkably, when challenged to chemical skin carcinogenesis, Loxl2-overexpressing mice increased tumor burden and malignant progression, while Loxl2-deficient mice exhibit the opposite phenotypes. Loxl2 levels in premalignant tumors negatively correlate with expression of epidermal differentiation markers and components of the Notch1 pathway. We show that LOXL2 is a direct repressor of NOTCH1. Additionally, we identify an exclusive expression pattern between LOXL2 and members of the canonical NOTCH1 pathway in human HNSCC. Our data identify for the first time novel LOXL2 roles in tissue homeostasis and support it as a target for SCC therapy.     

Mutational analysis of cutaneous squamous cell carcinomas and verrucal keratosis in patients taking BRAF inhibitors

B-RAF inhibitors (BRAFi) have been shown to improve rates of overall and progression-free survival in patients with stage IV metastatic melanoma positive for the BRAF V600E mutation. However, the main drawback is the development of verrucal keratosis (hyperkeratotic papules with verruca-like characteristics with benign histological findings) and cutaneous squamous cell carcinomas (cuSCC). We have found upstream mutations in RAS as well as PIK3CA in both verrucal keratosis and cuSCC. This suggests that verrucal keratosis is an early clinical presentation of cuSCC in patients on BRAFi.     

Detection and cloning of epidermal zinc-α2-glycoprotein cDNA and expression in normal human skin and in tumors

Zinc-α₂-glycoprotein (Znα₂gp) is almost ubiquitous in body fluids, and its antibody labels the corresponding secretory epithelia. We have found that Znα₂gp is also expressed in human epidermis. We cloned the Znα₂gp cDNA by screening our cDNA library, derived from epidermal keratinocytes, with a probe for prostate Znα₂gp. It had complete nucleic acid sequence homology with that from prostate, including the signal peptide. Just as Znα₂gp expression is higher in more differentiated breast tumors, so in skin tumors the highest mRNA levels occurred in the normal controls, the lowest in basal cell carcinomas (the least differentiated epidermal tumor type), and intermediate levels in squamous cell carcinomas and Merkel cell carcinomas. A similar increase in Znα₂gp gene expression with differentiation was observed when epidermal keratinocytes were cultured in media that varied in cellular maturation potential. J. Cell. Biochem. 67:216–222, 1997. © 1997 Wiley-Liss, Inc.     

Cytological diagnosis of basal cell carcinoma and actinic keratosis, using Papanicolaou and May–Grünwald–Giemsa stained cutaneous tissue smear

Objective:  Cytology may become the diagnostic method of choice with the advent of new non-invasive treatments for non-melanoma skin cancer, as the sampling technique for cytology entails little tissue disfiguration. The aim of this study was to compare and evaluate the diagnostic performance of scrape cytology using two different cytological staining techniques, and to evaluate additional touch imprint cytology, with that of histopathology of basal cell carcinoma (BCC) and actinic keratosis (AK).
Methods:  We investigated 50 BCC and 28 AK histologically verified lesions, from 41 and 25 patients, respectively. Two separate skin scrape samples and one touch imprint sample were taken from each lesion. The smears were stained with Papanicolaou (Pap) or May–Grünwald–Giemsa (MGG) stains. All cytological specimens were examined in random order by pathologists without knowledge of the histology. Cytodiagnostic results were compared with the histopathological report.
Results:  Scrape cytodiagnosis agreed with histopathology in 48 (Pap) and 47 (MGG) of the 50 BCC cases, and in 26 of 28 (Pap) and 21 of 26 (MGG) AK cases, yielding sensitivities of 96%, 94%, 93% and 81%, respectively. No significant difference in sensitivity between the two staining methods was found but a trend towards higher Pap sensitivity for AK was noted ( P  =   0.10). Touch imprint cytology confirmed histopathology in 38 of the 77 cases of BCC and AK.
Conclusion:  Cytological diagnosis with either Pap or MGG stain for BCC and AK is reliable, and differentiates well between BCC and AK. Imprint cytology proved to be non-diagnostic in half of the examined cases.     

YAP and TAZ are essential for basal and squamous cell carcinoma initiation

YAP and TAZ are key downstream regulators of the Hippo pathway, regulating cell proliferation and differentiation. YAP and TAZ activation has been reported in different cancer types. However, it remains unclear whether they are required for the initiation of major skin malignancies like basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). Here, we analyze the expression of YAP and TAZ in these skin cancers and evaluate cancer initiation in knockout mouse models. We show that YAP and TAZ are nuclear and highly expressed in different BCC types in both human and mice. Further, we find that cells with nuclear YAP and TAZ localize to the invasive front in well‐differentiated SCC, whereas nuclear YAP is homogeneously expressed in spindle cell carcinoma undergoing EMT. We also show that mouse BCC and SCC are enriched for YAP gene signatures. Finally, we find that the conditional deletion of YAP and TAZ in mouse models of BCC and SCC prevents tumor formation. Thus, YAP and TAZ are key determinants of skin cancer initiation, suggesting that targeting the YAP and TAZ signaling pathway might be beneficial for the treatment of skin cancers.     

Sonic hedgehog opposes epithelial cell cycle arrest.

Stratified epithelium displays an equilibrium between proliferation and cell cycle arrest, a balance that is disrupted in basal cell carcinoma (BCC). Sonic hedgehog (Shh) pathway activation appears sufficient to induce BCC, however, the way it does so is unknown. Shh-induced epidermal hyperplasia is accompanied by continued cell proliferation in normally growth arrested suprabasal cells in vivo. Shh-expressing cells fail to exit S and G2/M phases in response to calcium-induced differentiation and also resist exhaustion of replicative growth capacity. In addition, Shh blocks p21(CIP1/WAF1)-induced growth arrest. These data indicate that Shh promotes neoplasia by opposing normal stimuli for epithelial cell cycle arrest.     

Osteopontin expression in coculture of differentiating rat fetal skeletal fibroblasts and myoblasts

Summary Skeletal fibroblasts in vitro can acquire myofibroblast phenotypes by the development of biochemical and morphological features, mainly the expression of alpha-smooth-muscle actin (α-SMA). Myogenic differentiation is a central event in skeletal muscle development, and has commonly been studied in vitro in the context of skeletal muscle development and regeneration. Controlling this process is a complex set of interactions between myoblasts and the extracellular matrix. Osteopontin (OPN) is an acidic, phosphorylated matrix protein that contains an Arg-Gly-Asp (RGD) cell attachment sequence and has been identified as an adhesive and migratory substrate for several cell types. The aim of this study was to investigate osteopontin expression during the differentiation of skeletal fibroblasts into myofibroblasts and during myogenesis in a coculture model. Fibroblasts and myoblasts were obtained from skeletal muscle of 18-d-old Wistar strain rat fetuses by enzymatic dissociation. At 1 and 9 d, cocultures were immunolabeled, and the cells were also separately subjected to Western blotting to analyze OPN expression. Our data using confocal microscopy showed that myoblasts displayed a strong staining for OPN and that this labeling was maintained after myotube differentiation. Conversely, during fibroblast differentiation into myofibroblasts, we observed a significant increase in OPN expression. The results obtained by immunolabeling were confirmed by Western blotting. We suggest that OPN is important mainly during early stages of myogenesis, facilitating myoblast fusion and differentiation, and that the increased expression of OPN in myofibroblasts might be related to its effects as a key cytokine regulating tissue repair and inflammation.     

Differentiation of normal and tumoral human keratinocytes cultured on dermis: Reconstruction of either normal or tumoral architecture

Summary Normal human keratinocytes isolated from skin and squamous carcinoma cells established from a human tumor (TR146 cell line) both exhibit limited morphologic differentiation when they are grown on conventional plastic dishes. However, when they are seeded on human de-epidermized dermis and cultured at the air-liquid interface, they are able to reform an epithelium having the morphology of the tissue of origin (i.e. skin or squamous carcinoma). The distribution in such reconstructed tissues of differentiation markers such as bullous pemphigoid antigen, 67K keratin, involucrin, membrane-bound transglutaminase, and filaggrin was very similar to their distribution in normal skin and squamous carcinoma specimens, respectively. The degree of differentiation is for both cell types extremely sensitive to culture conditions such as retinoic acid concentration, emersion of the cultures, etc. These results show that subcultured normal or tumoral keratinocytes are able to recover their specific morphogenetic potential when cultured in an environment close to their in vivo situation.     

Trends in incidence and survival analysis in non-melanoma skin cancer from 1994 to 2012 in Girona,Spain: A population-based study

IntroductionWe present an epidemiological study focused on Non-melanoma skin cancers (NMSC), including squamous cell carcinoma (SCC), basal cell carcinoma (BCC), Merkel cell carcinoma (MCC), dermatofibrosarcoma protuberans (DFS) and adnexal and skin appendages neoplasm (ASAN), a neoplasm understudied in cancer registries.Material and methodsWe analyze trends of incidence and survival of NMSC registered with the Cancer Registry of Girona, Spain.ResultsWe found 14389 cases of NMSC, accounting 3,474 SCC, 10729 BCC, 33 MCC, 61 DFSP and 71 ASAN. Incidence increased significantly in SCC and BCC with annual percentage of change of 1.6 and 1.5, respectively, but not in MCC, DFS or ASAN. Five-year relative survival for both sexes was 90.1% in SCC, 99.8% in BCC, 44.2% in MCC, 93.7% in DFS and 84% in ASAN.ConclusionsOur study confirms the increasing incidence and good survival of SCC and BCC and enhances knowledge on the epidemiology of the less incidental NMSC.     

Chronic UVR Causes Increased Immunostaining of CD44 and Accumulation of Hyaluronan in Mouse Epidermis

Chronic intense UV radiation is the main cause of epidermal tumors. Because hyaluronan (HA), a large extracellular polysaccharide, is known to promote malignant growth, hyaluronan expression was studied in a model in which long-term UV radiation (UVR) induces epidermal tumors. Mouse back skin was exposed three times a week for 10.5 months to UVR corresponding to one minimal erythema dose, processed for histology, and stained for hyaluronan and the hyaluronan receptor CD44. This exposure protocol caused epidermal hyperplasia in most of the animals; tumors, mainly squamous cell carcinomas (SCCs), were found in ~20% of the animals. Specimens exposed to UVR showed increased hyaluronan and CD44 staining throughout the epidermal tissue. In hyperplastic areas, hyaluronan and CD44 stainings correlated positively with the degree of hyperplasia. Well-differentiated SCCs showed increased hyaluronan and CD44 staining intensities, whereas poorly differentiated tumors and dysplastic epidermis showed areas where HA and CD44 were locally reduced. The findings indicate that HA and CD44 increase in epidermal keratinocytes in the premalignant hyperplasia induced by UV irradiation and stay elevated in dysplasia and SCC, suggesting that the accumulation of hyaluronan and CD44 is an early marker for malignant transformation and may be a prerequisite for tumor formation.     

Differentiation of cultured human keratinocytes: Effect of culture conditions on lipid composition of normal vs. malignant cells

Summary Differentiation in keratinocytes can be experimentally modulated by changing the culture conditions. When cultured under conventional, submerged conditions, the extent of cellular differentiation is reduced in the presence of low calcium medium and is enhanced in medium containing physiologic calcium concentrations. Moreover cultures grown at the air-medium interface or on a dermal substrate, or both, differentiate even further. Herein we report the effect of culture conditions on lipid composition in normal human keratinocytes and three squamous carcinoma cell (SCC) lines that vary in their capacity to differentiate as assessed by cornified envelope formation. Under submerged conditions, the total phospholipid content was lower, triglyceride content higher, and phospholipid: neutral lipid ratio lower in direct correlation to the degree of differentiation in these cultures. When grown at the air-medium interface on the-epidermized dermis, evidence of further morphologic differentiation was found only for well-differentiated SCC cells and normal keratinocytes. Similarly, the phospholipid content remained high in poorly differentiated SCC cells and it, decreased modestly in well-differentiated SCC cells and markedly in normal keratinocytes. In all cell lines the triglyceride content was increased and cholesterol content decreased when compared to parallel submerged cultures, but these differences were most pronounced in well-differentiated cell lines. Acylceramides and acylglucosylceramides were found only in normal keratinocytes and only under the most differentiation-enhancing conditions. These studies demonstrate differentiation-related changes in the lipid content of both normal and neoplastic keratinocytes. This work was supported in part by NATO Scientific Award (RG 8510056).     

A histochemical comparison of methyl green-pyronin,and hematoxylin and eosin for detecting apoptotic cells in oral squamous cell carcinoma,oral leukoplakia,oral submucous fibrosis and normal oral mucosa

Abstract

Analysis of apoptotic cells in oral pathological states could be useful for determining the rates of tissue turnover, which would help determine prognosis. The use of histochemical stains such as hematoxylin and eosin (H & E) and methyl green-pyronin (MGP) can provide a simple and cost-effective method for detecting apoptotic cells. We compared the efficacy of MGP and H & E for detecting apoptotic cells in oral squamous cell carcinoma (OSCC), oral leukoplakia (OL), oral submucous fibrosis (OSMF) and normal oral mucosa (NOM). Ten cases each of OSCC, OSMF, OL and NOM were retrieved from the archives and two serial sections were stained, one with H & E and the other with MGP. Apoptotic cells were identified at 100 x magnification and the apoptotic index was calculated. Apoptotic cells were distinguished more readily in MGP stained sections than in those stained with H & E. Also, the apoptotic cell count was greater in OSCC compared to OL, OSMF and NOM. We concluded that MGP staining can be used as a routine, cost-effective method for detecting apoptotic cells.     

The treatment with differentiation- and apoptosis-inducing agent, vesnarinone, of a patient with oral squamous cell carcinoma

A patient with histopathological recurrent oral cancer with well-differentiated squamous cell carcinoma, was treated with differentiation- and apoptosis-inducing agent, vesnarinone, per os at a dose of 180 mg/day for 56 days and then at a dose of 60 mg/day for 93 days. The vesnarinone administration caused complete remission of the tumour. It has been found by immunohistochemical staining and PCR-SSCP analysis that the recurrent tumour has wild type p53 gene and relative high level of LeY expression as well as DNA fragmentation in the cancer cells, as assessed by nick-end labelling. These findings suggest that the cure of oral squamous cell carcinoma observed in this case might be associated with induction of differentiation and apoptosis of cancer cells by vesnarinone.     

NUMB在口腔白斑和鳞癌中的表达变化及意义

目的:研究NUMB在口腔白斑和鳞癌中的表达及意义。方法:采用免疫组织化学法检测88例口腔正常黏膜、口腔白斑及口腔鳞癌石蜡包埋组织中NUMB蛋白的表达。结果:NUMB在口腔正常黏膜(95.7%)、口腔白斑(75%)及口腔鳞癌(4.7%)中均有阳性表达但是表达频率依次降低。NUMB在各个组中的阳性表达率具有统计学差异(P0.05)。结论NUMB阳性表达率随口腔组织恶性程度增高而减少的趋势提示该基因可能在口腔正常黏膜到口腔白斑再到口腔鳞癌的转化中起作用,NUMB有可能成为早期发现癌变的分子生物学标志之一。