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.     

Human papillomaviruses in oncogenesis

Papillomaviruses, long associated with benign skin tumors, have been linked more recently to human cancers, particularly to cervical carcinoma. Molecular analysis of the virus has identified the transforming gene and its regulation by both viral and cellular trans:-acting factors. This viral regulatory mechanism is altered in carcinomas. However, lack of progress in developing an in vitro system has hampered investigation of the viral life cycle and the biology of the virus--host: cell interaction.     

NOREPINEPHRINE-STIMULATED BREAKDOWN OF TRIPHOSPHOINOSITIDE OF RABBIT IRIS SMOOTH MUSCLE: EFFECTS OF SURGICAL SYMPATHETIC DENERVATION AND IN VIVO ELECTRICAL STIMULATION OF THE SYMPATHETIC NERVE OF THE EYE

Abstract— Paired iris smooth muscles from rabbits were prelabelled either in vitro by incubation for 30 min at 37°C in an iso-osmotic salt medium containing glucose, inositol, cytidine and ³²Pi, or in vivo by administration of the isotope intracamerally into each eye 1 h before death. One of the pair was then incubated at 37°C for 10 min in an unlabelled medium containing 10 mm of 2-deoxyglucose and the other was incubated in the presence of norepinephrine (NE) or other adrenergic agents. Triphosphoinositide (TPI) was found to contain more ³²P than any other phospholipid (almost 39% of total lipid radioactivity) in both the in vitro and in vivo experiments. NE (50 μm ) increased the loss of ³²P from TPI (the TPI effect') by 28–30% in the ³²P-labelled muscle. The TPI effect was accompanied by a significant increase in ³²P labelling of phosphatidic acid (PA) and phosphatidylinositol (PI) but not phosphatidylcoholine. In this tissue the TPI effect was found to be mediated through α-adrenergic receptors. At 14 days after surgical sympathetic denervation, incorporation of ³²P into phospholipids of the denervated muscle increased by an average of 6% over that of the normal muscle. The increase in TPI, PI and PA was 7%, 4% and 9% of that of the control respectively. There was little change in phospholipid content of the denervated muscle. The increase in sensitivity to NE (12.5 μm ) caused by denervation produced about 18% increase in the TPI effect and a 25% increase in the ³²P labelling of PA, but not PI. In view of our previous findings on the requirement of the TPI effect for Ca²⁺, this observation could suggest that an increase in Ca²⁺ influx, following the interaction between the neurotransmitter and its receptor could stimulate TPI-phosphodiesterase, thus leading to increased PA via increased diglyceride. This denervation-induced supersensitivity to NE appears to be postsynaptic in nature. ³²Pi was injected intracamerally into each eye 1 h before electrical stimulation of one of the sympathetic trunks. After stimulation for 30 min there was a significant loss of ³²P from TPI and a significant increase in the labelling of PI and PA of the stimulated muscle. It is concluded that TPI and its enzymes could play an important role in neurotransmission at the neuromuscular junction of smooth muscle.     

Acute cytotoxicity testing with cultured human lung and dermal cells

Summary An extensive in vitro study with cultured cells was conducted to test the basal cytotoxicity theory. This theory suggests that most chemical injury, at least in vitro, is a manifestation of one or more insults to the basic cellular structures and functions common to mammalian cells. This accounts for the similarity of results in multilaboratory studies. Human fetal lung fibroblasts (HFL1), and human skin fibroblasts (WS1, Detroit551) were studied in culture to evaluate their potential to screen for cytotoxicity. Confluent monolayers were incubated in the absence or presence of increasing concentrations of test chemicals for 24 h, and the MTT assay was used to assess toxicity. Inhibitory concentrations were extrapolated from concentration-effect curves after linear regression analysis. Twenty-nine chemicals were tested with each cell line and the cytotoxicity data compared to rodent and human lethal concentrations. The data suggest that the experimental IC₅₀ values are as accurate predictors of human toxicity as equivalent toxic blood concentrations derived from rodent LD₅₀s. In addition, lung and skin fibroblasts revealed no significant differences among the three cell lines. The results support the conclusion that finite cell lines of human origin have the potential for screening chemicals for human toxicity. In combination with previously published reports, the data suggest that a basal cytotoxic phenomenon may explain the similarity of results among different human cell lines.     

UV-specific mutations of the human patched gene in basal cell carcinomas from normal individuals and xeroderma pigmentosum patients

Germline mutations of the human patched gene, PTCH, are responsible for the nevoid basal cell carcinoma (NBCC) syndrome or Gorlin's syndrome, characterized by multiple skin cancers, internal cancers and severe developmental abnormalities. The patched gene codes for a developmental regulator protein implicated in the sonic hedgehog (SHH) signalling pathway which plays an important role in oncogenic transformation. Patched exhibits tumor suppression function and has been shown to be mutated in skin cancers isolated from DNA repair-proficient patients or from xeroderma pigmentosum (XP), a DNA repair-deficient syndrome.We have reviewed and analyzed in detail the different mutation spectra found on the PTCH gene in these various models. The type and distribution of mutations are quite different between germline, sporadic and XP cancers. Among the germline alterations, there is a preponderance (70%) of rearrangements compared to other tumour types analysed where less than 30% of rearrangements is observed. Typical UV-induced mutations of the patched gene are found prominently in XP basal cell carcinomas (BCCs) and in particular, a significantly higher level (63%) of the UV signature tandem mutations is found compared to sporadic BCC (11%). The location of mutations along the PTCH protein delineates several important functional domains implicated in the biology of this transmembrane receptor.     

Interaction of triosephosphate isomerase from Staphylococcus aureus with plasminogen

Triosephosphate isomerase (TPI; EC 5. 3. 1. 1) displayed on the cell surface of Staphylococcus aureus acts as an adhesion molecule that binds to the capsule of Cryptococcus neoformans, a fungal pathogen. This study investigated the function of TPI on the cell surface of S. aureus and its interactions with biological substances such as fibronectin, fibrinogen, plasminogen, and thrombin were investigated. Binding of TPI to plasminogen was demonstrated by both surface plasmon resonance analysis and Far‐Western blotting. It is suggested that lysine residues contribute to this binding because the interaction was inhibited by ?‐aminocaproic acid. Activation of plasminogen to plasmin by staphylokinase or tissue plasminogen activator decreased in the presence of TPI, whereas TPI was degraded by plasmin. In other experiments, intact S. aureus cells had the ability to both increase and decrease plasminogen activation depending on the number of cells. Several molecules expressed on the surface of S. aureus were predicted to interact with plasminogen, resulting in its increased or decreased activation. These findings indicate that S. aureus sometimes localizes and sometimes disseminates in the host, depending on the molecules expressed under various conditions.     

Cell culture aging

Summary Cellular research in aging has been stimulated by the observation that human diploid cells have a limited number of cell divisions in culture. This loss of cellular proliferation (in vitro senescence) has been extensively studied by biochemical, clonal, and genetic analysis. Studies of human skin fibroblast cultures have revealed thatin vitro senescense is related toin vivo human cellular aging. Recently differentiated cells have been proposed for aging studies. These cells may provide additional information on aging since alterations ofin vitro cellular functions may be related to thein vivo behavior of specific differented cell types.     

Excision margins for nonmelanotic skin cancer

Scientific evidence for advisable excision margins for nonmelanotic skin carcinoma is poorly documented. Recommended excision margins vary from 2 to 15 mm. A prospective study was performed on 150 skin lesions excised over a 9-month period in an outpatient facility at the authors' institution. Primary nonmelanotic skin lesions were clinically diagnosed as either basal cell carcinoma (nodular, superficial, infiltrating, or sclerosing) or squamous cell carcinoma (well, moderately, or poorly differentiated). Macroscopic surgical excision margins were individually assessed, measured, and excised. Histopathologic analysis was then independently performed to determine the correct diagnosis and to measure the actual microscopic lateral and deep excision margins.Sixty-one percent of lesions were basal cell carcinoma, 25 percent were squamous cell carcinoma, and 15 percent were benign or premalignant. Diagnostic accuracy was 81 percent for basal cell and 59 percent for squamous cell carcinoma. The average diameter of the basal cell carcinoma was 12.1 mm; 47 percent of these lesions had a diameter of less than 10 mm. The average diameter of the squamous cell carcinoma was 16.9 mm; 26 percent of these lesions had a diameter of less than 10 mm. The mean surgical margin was 4.2 mm (3.2 mm adjusted for shrinkage), whereas the mean microscopic lateral margin was 3.4 mm. Overall, complete excision was achieved for 98 percent of basal cell carcinoma and 100 percent of squamous cell carcinoma. The raw data were analyzed to assess the suitability of 1-, 2-, 3-, or 4-mm surgical excision margins. A 4-mm surgical margin would give a microscopic lateral margin beyond one microscopic high-power field (0.5 mm) in 96 percent of cases of basal cell carcinoma and in 97 percent of cases of squamous cell carcinoma.The authors recommend a 4-mm surgical margin as the optimal treatment for skin lesions clinically diagnosed as basal cell or squamous cell carcinoma that are suitable for excision in an outpatient facility. Well-demarcated lesions, such as a nodular basal cell carcinoma, may be excised with a 3-mm margin.     

Radiosensitization by Thymidine Phosphorylase Inhibitor in Thymidine Phosphorylase Negative and Overexpressing Bladder Cancer Cell Lines

TAS-102 (trifluorothymidine [TFT] and thymidine phosphorylase inhibitor [TPI] in a molar ratio of 1:0.5) has activity in 5-fluorouracil resistant colon cancer. TPI is added to increase TFT's bioavailability. TFT has a dual mechanism of action by inhibiting thymidylate synthase and by its incorporation into DNA. Interesting radiosensitizing effects of TPI were recently reported. The aim of our study was to determine whether TP expression would affect radiosensitivity and to characterize the effect of TPI. Two bladder cancer cell lines RT112 (TP negative) and RT112/TP (TP overexpression) were tested for drug sensitivity and radiosensitivity (clonogenic assay), with and without TFT and/or TPI. Expression of γ H2AX was used as marker for DNA damage. RT112 cells were not more sensitive to TFT then RT112/TP cells. TPI alone did not inhibit cell growth of RT112 even at 100 μM, but inhibited that of RT112/TP by 27%. In both RT112 and RT112/TP cells 10 μM TPI did not or slightly affect radiosensitivity, but 100 μM TPI alone enhanced the radiation response (p <.05). TFT alone at 1 μM and in combination with 10 μM TPI did not affect the radiation response of both cell lines. TPI alone induced expression of ?H2AX, which was increased in combination with radiation. In conclusion, TPI enhanced radiosensitivity at high concentrations, independent of TP expression, while TFT and TPI at a low concentration did not affect the radiosensitivity of RT112 and RT112/TP cell lines.     

Expression patterns of α2,3-Sialyltransferase I and α2,6-Sialyltransferase I in human cutaneous epithelial lesions

Skin tumors have become one of the most common cancers in the world and their carcinogenesis is frequently associated with altered glycosylation patterns. The aberrant sialylation, a type of glycosylation, can mediate pathophysiological key events during various stages of tumor progression, including invasion and metastasis. Sialyltransferases play a key role in a variety of biological processes, including cell-cell communication, cell-matrix interaction, adhesion, and protein targeting. In this study, it was evaluated the expression of ST3Gal I and ST6Gal I in cutaneous epithelial lesions that include actinic keratosis (n=15), keratoacanthoma (n=9), squamous cell carcinoma (n=22) and basal cell carcinoma (n=28) in order to evaluate if sialyltransferases expression is different in premalignant and in malignant tumors. The expression of ST3Gal I was observed in actinic keratosis (53%), keratoacanthoma (78%), squamous cell carcinoma (73%) and basal cell carcinoma (32%) with statistic differences between basal cell carcinoma and keratoacanthoma (P=0.0239) and basal cell carcinoma and squamous cell carcinoma (P=0.0096); for ST6Gal I, cytoplasmic expression was noted in actinic keratosis (40%), heterogeneous and cytoplasmic expression was noted in keratoacanthoma (67%), squamous cell carcinoma (41%) and basal cell carcinoma (7%) with statistic differences between basal cell carcinoma and squamous cell carcinoma (P=0.0061) and basal cell carcinoma and keratoacanthoma (P=0.0008). In summary, our results showed that the high expression of ST3Gal I and ST6Gal I, in skin tumors, is associated with tumors with greater potential for invasion and metastasis, as in the case of squamous cell carcinoma, and this may be related to their behavior.Key words: sialic acid, α2,3-sialyltransferases, α2,6-sialyltransferases, basal cell carcinoma, squamous cell carcinoma, actinic keratosis, keratoacanthoma     

Immunohistochemical analysis of DNA mismatch repair enzyme hMSH-2 in normal human skin and basal cell carcinomas

We have analysed the expression and distribution of the DNA mismatch repair enzyme hMSH-2 in normal skin and basal cell carcinomas. hMSH-2 protein was investigated immunohistochemically (normal human skin: n=10; basal cell carcinomas: n=16) on frozen sections using a highly sensitive streptavidin–peroxidase technique and a specific mouse monoclonal antibody (clone FE11). In normal human skin, we found nuclear immunoreactivity for hMSH-2 in epidermal keratinocytes of the basal and first 1–3 suprabasal cell layers. All basal cell carcinomas analysed revealed strong nuclear imunoreactivity that was pronounced in peripheral tumour cells and cells of the palisade. Expression of hMSH-2 protein was consistently and strongly upregulated in tumour cells of the carcinomas as compared to adjacent unaffected epidermis or epidermis of normal human skin. Twelve of the sixteen carcinomas analysed revealed no visual correlation in comparing the labelling patterns for hMSH-2 with the labelling pattern for the proliferation marker Ki-67. Our findings indicate that (a) hMSH-2 is expressed in human epidermal keratinocytes, predominantly in lower cell layers of the viable epidermis; (b) expression of hMSH-2 protein is strongly upregulated in basal cell carcinomas as compared to unaffected epidermis; (c) the level of hMSH-2 proteins in the carcinomas is not exclusively regulated by the proliferative activity of these tumour cells; (d) inactivating mutations of the hMSH-2 gene may in the carcinomas not be involved in the carcinogenesis or microsatellite instability secondary to replication errors; (e) expression of hMSH-2 may be of importance for the genetic stability of basal cell carcinomas in vivo.     

Localization by scanning immunoelectron microscopy of triosephosphate isomerase,the molecules responsible for contact‐mediated killing of Cryptococcus,on the surface of Staphylococcus

In our previous studies, TPI were found to be the molecules responsible for contact‐killing of C. neoformans by S. aureus cells. Since TPI is a glycolytic protein that functions in the cytoplasm, evidence that TPI is present on the surface of S. aureus was required. In the present study, the presence of TPI on the cell surface of S. aureus was demonstrated by agglutination test and scanning immunoelectron microscopy. Furthermore, TPI was found to be present at a lower density than protein A/G molecules on the surface of S. aureus.     

Localization of triphosphoinositide in the cochlea

Summary Triphosphoinositide (TPI) has been demonstrated to be a receptor for aminoglycosides in the cochlea and may regulate ionic permeability by its binding with Ca⁺⁺. This phospholipid was localized by a protein A-gold technique in the cochlea at the electronmicroscopic level. TPI was prepared by a neomycin column and antibodies to it were raised in rabbits. The antibody used in this study reacted virtually only to TPI among the tested lipids. TPI was localized mainly at stereocilia, cuticular plates, head plates of Deiter's cells, plasma membrane, and mitochondria of various cells in the organ of Corti. In the vascular stria, TPI was found mainly at the plasma membrane of basal infoldings of the marginal cells. Possible physiological and pathophysiological roles of TPI in the cochlea are briefly discussed.     

In vivo measurements of diffuse reflectance and time‐resolved autofluorescence emission spectra of basal cell carcinomas

We present a clinical investigation of diffuse reflectance and time‐resolved autofluorescence spectra of skin cancer with an emphasis on basal cell carcinoma. A total of 25 patients were measured using a compact steady‐state diffuse reflectance/fluorescence spectrometer and a fibre‐optic‐coupled multispectral time‐resolved spectrofluorometer. Measurements were performed in vivo prior to surgical excision of the investigated region. Singular value decomposition was used to reduce the dimensionality of steady state diffuse reflectance and fluorescence spectra. Linear discriminant analysis was then applied to the measurements of basal cell carcinomas (BCCs) and used to predict the tissue disease state with a leave‐one‐out methodology. This approach was able to correctly diagnose 87% of the BCCs. With 445 nm excitation a decrease in the spectrally averaged fluorescence lifetime was observed between normal tissue and BCC lesions with a mean value of 886 ps. Furthermore, the fluorescence lifetime for BCCs was lower than that of the surrounding healthy tissue in all cases and statistical analysis of the data revealed that this decrease was significant (p = 0.002). (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Human keratinocytes cultured on collagen gels form an epidermis which synthesizes bullous pemphigoid antigens and α2β1 integrins and secretes laminin, type IV collagen, and heparan sulfate proteoglycan at the basal cell surface

Single cell suspensions of human keratinocytes when seeded onto floating three-dimensional gels constructed with type I collagen form a tissue resembling epidermis. These morphogenetic events occur in a serum-free environment in the absence of fibroblasts. Light and transmission electron microscopy show that cells form a basal layer plus suprabasilar cell layers corresponding to the stratum spinosum, stratum granulosum, and stratum corneum. The suprabasilar keratinocyte layers show morphologies which resemble intact skin in which cells are connected by desmosomes and contain intermediate filaments and keratohyalin-fillagrin granules. The basal cell layer differs from skin in vivo in that there is no connection to a basement membrane via hemidesmosomes. Cells in the basal layers are polarized as evidenced by the secretion of type IV collagen, heparan sulfate proteoglycans, and laminin at the cell membrane interface with the collagen gel. These proteins are not organized into a cytological basement membrane. Bullous pemphigoid antigen, a protein component of hemidesmosomes, is synthesized by basal keratinocytes, but like the basement membrane proteins it is not incorporated into a definable cytological structure. Keratinocytes in the basal and suprabasilar layers also synthesize α2β1 integrins. The mechanisms of keratinocyte adhesion to the gel may be through the interactions of this cell surface receptor with laminin and type IV collagen synthesized by the cell and/or direct interactions between the receptor and type I collagen within the gel. This in vitro experimental system is a useful model for defining the molecular events which control the formation and turnover of basement membranes and the mechanisms by which keratinocytes adhere to type I collagen when sheets of keratinocytes are used clinically for wound coverage.     

Expression of the HB9 homeobox gene concomitant with proliferation accompanying epidermal stratification during development of chick embryonic tarsometatarsal skin

A homeobox gene, HB9, has been isolated from the tarsometatarsal skin of 13-day-old chick embryos using a degenerate RT-PCR-based screening method. In situ hybridization analysis revealed that, during development of chick embryonic skin, the HB9 gene was expressed in epidermal basal cells of the placodes, but not in those of interplacodes, and in the dermal cells under the placodes at 9 days before addition of an intermediate layer by proliferation of the basal cells in the placodes. With the onset of epidermal stratification, the direction of the basal cell mitosis changed, with the axis becoming vertical to the epidermal surface. Placodes and interplacodes form outer and inner scales, respectively, after they have elongated distally (Tanaka S, Kato Y (1983b) J Exp Zool 225: 271–283). During scale ridge elongation at 12–15 days, HB9 was strongly expressed in the epidermis of the outer scale face, where the cell proliferation is more active than in the epidermis of the inner scale face; hence, stratification of the outer scale face is more prominent than that of the inner scale face. After 16 days, when mitotic activity in the epidermal basal cells decreases and the thickness of the epidermis is maintained at a constant level, the HB9 expression decreases with the onset of epidermal keratinization. These results suggest that HB9 may be involved in the proliferation of the epidermal basal cells that accompanies epidermal stratification.     

Elevated nuclear localization of glycolytic enzyme TPI1 promotes lung adenocarcinoma and enhances chemoresistance

Increased glycolysis is a hallmark of tumor, which can provide tumor cells with energy and building blocks to promote cell proliferation. Recent studies have shown that not only the expression of glycolytic genes but also their subcellular localization undergoes a variety of changes to promote development of different types of tumors. In this study, we performed a comprehensive analysis of glycolysis and gluconeogenesis genes based on data from TCGA to identify those with significant tumor-promoting potential across 14 types of tumors. This analysis not only confirms genes that are known to be involved in tumorigenesis, but also reveals a significant correlation of triosephosphate isomerase 1 (TPI1) with poor prognosis, especially in lung adenocarcinoma (LUAD). TPI1 is a glycolytic enzyme that interconverts dihydroxyacetone phosphate (DHAP) to glyceraldehyde 3-phosphate (GAP). We confirm the upregulation of TPI1 expression in clinical LUAD samples and an inverse correlation with the overall patient survival. Knocking down of TPI1 in lung cancer cells significantly reduced cell migration, colony formation, and xenograft tumor growth. Surprisingly, we found that the oncogenic function of TPI1 depends on its translocation to cell nucleus rather than its catalytic activity. Significant accumulation of TPI1 in cell nucleus was observed in LUAD tumor tissues compared with the cytoplasm localization in adjacent normal tissues. Moreover, nuclear translocation of TPI1 is induced by extracellular stress (such as chemotherapy agents and peroxide), which facilitates the chemoresistance of cancer cells. Our study uncovers a novel function of the glycolytic enzyme TPI1 in the LUAD.Subject terms: Non-small-cell lung cancer, Cell biology