The role of the sonic hedgehog signaling pathway in epidermal homeostasis

Non-melanoma skin cancer is the most frequently diagnosed malignancy in the United States of which basal cell carcinoma (BCC) accounts for 65%. It has recently been determined that deregulation of the sonic hedgehog (shh) pathway leads to the development of BCC. Shh, gli-1, gli-2 gli-3, ptc and smo are overexpressed in BCC and overexpression of these genes in the epidermis results in formation of BCC-like tumors. Despite these observations, the mechanisms by which the pathway controls epidermal homeostasis and the development of the malignant phentotype are unknown. This study assessed the role of the shh pathway in epidermal homeostasis through regulation of apoptosis and differentiation. ^ The anti-apoptotic protein, bcl-2 is overexpressed in BCC, however transcriptional regulators of bcl-2 in the epidermis are unknown. Transient transfection of primary keratinocytes with gli-1 resulted in an increase of bcl-2 expression. Database analysis revealed seven candidate gli binding sites on the bcl-2 promoter. Cotransfection of increasing amounts of gli-1 in keratinoycytes resulted in a corresponding dose-dependent increase in bcl-2 promoter luciferase activity. An N-terminal mutant of gli-3 inhibited gli-1 transactivation of the bcl-2 promoter. The region −428 to −420 was found to be important for gli-1 regulation through gel shift, luciferase assays and site-directed mutagenesis. ^ In order to assess the ability of the shh pathway to regulate keratinocyte differentiation, HaCaT keratinocytes overexpressing sonic hedgehog, were grown in organotypic raft culture. Overexpression of shh induced a basal cell phenotype compared to vector control, as evidenced by transmural staining of cytokeratin 14 and altered Ki67 staining. Shh also induced keratinocyte invasion into the underlying collagen. This was associated with increased phosphorylation of EGFR, jnk and raf and increased expression of c-jun, mmp-9 and Ki67. Interestingly, shh overexpression in HaCaTs did not induce the typical downstream effects of shh signaling, suggesting a gli-independent mechanism. Sonic hedgehog's ability to induce an invasive phenotype was found to be dependent on activation of the EGF pathway as inhibition of EGFR activity with AG1478 and c-225 was able to reduce the invasiveness of HaCaT shh keratinocytes, whereas treatment with EGF augmented the invasiveness of the HaCaT shh clones. ^ These studies reveal the importance of the sonic hedgehog pathway in epidermal homeostasis by regulation of apoptosis through bcl-2, and control of keratinocyte differentiation and invasion through activation of the EGF pathway. They further suggest potential mechanisms by which deregulation of the shh pathway may lead to the development of the malignant phenotype. ^

Calcium mobilization from the endoplasmic reticulum to the mitochondria during apoptosis in prostate cancer

Previous studies have implicated Ca2+ fluxes in the control of apoptosis but their exact roles in regulating the process remain obscure. Because Ca2+ can serve as a signal for cytochrome c release from isolated mitochondria, we hypothesized that alterations in intracellular Ca2+ compartmentalization might serve as a release signal in whole cells undergoing apoptosis. Exposure of human PC-3 prostate adenocarcinoma cells to staurosporine or DNA damaging agent (doxorubicin) but not to anti-Fas antibody led to early release of Ca2+ from the endoplasmic reticulum and subsequent accumulation of Ca2+ within mitochondria. Both events were blocked in cells stably transfected with Bcl-2 but were not affected by treatment with the pancaspase inhibitor, zVADfmk. The effects of staurosporine were associated with re-localization of Bax from the cytosol to both endoplasmic reticular and mitochondrial membranes. Neither ER Ca 2+ pool depletion nor mitochondrial Ca2+ uptake were observed in DU-145 cells that possess a frameshift mutation in the Bax gene unless wild-type Bax was restored via adenoviral gene transfer. Cytochrome c release and downstream features of apoptosis were attenuated by treatment with an inhibitor of mitochondria) Ca2+ uptake (RU-360). Although, direct pharmacological ER Ca2+ pool emptying in cells treated with thapsigargin did not lead to early cytochrome c release, pretreatment of cells with staurosporine dramatically sensitized mitochondria to thapsigargin-induced cytochrome c release. Together, our data demonstrate that ER-to-mitochondrial Ca2+ fluxes promote cytochrome c release and apoptosis in cells exposed to some (but not all) pro-apoptosic stimuli. ^

Effects of exogeneous p53 on growth suppression, apoptosis, and differentiation in oral cancer cell lines

The p53 gene is known to be one of the most commonly mutated genes in human cancers. Many squamous cell carcinomas of the head and neck (SCCHNs) have been shown to contain nonfunctional p53 as well. The use of p53-mediated gene therapy to treat such cancers has become an intensive area of research. Although there have been varied treatment responses to p53 gene therapy, the role that endogenous p53 status plays in this response has not been thoroughly examined. Because of this, the hypothesis of this study examined the role that the endogenous p53 status of cells plays in their response to p53 gene therapy. To test this, an adenoviral vector containing p53 (p53FAd) was administered to three squamous cell carcinoma lines with varied endogenous p53. The SCC9 cell line demonstrates no p53 protein expression, the SCC4 cell line displays overexpression of a mutant p53 protein, and the 1986LN cell line displays low to no expression of wild-type p53 protein as a consequence of human papillomavirus infection. After treatment with p53FAd, the cells were examined for evidence of exogenous p53 expression, growth suppression, alterations in cellular proteins, G1 growth arrest, apoptosis, and differentiation state. Each cell line exhibited exogenous p53 protein. Growth suppression was seen most prominently in the SCC9 cells, to some extent in the 1986LN cells, and little was seen with the SCC4 cells. WAF1/p21 protein was induced in all three cell lines, while PCNA, bcl-2, and bax expression was not significantly affected in any of the lines. Apoptosis developed first in SCC9 cells, next in 1986LN cells, with little seen in the SCC4 cells. The SCC9 line was the only line to show significant GI growth arrest. No significant differences were observed in the overall expression of differentiation markers, aside from increased keratin 13 mRNA levels in all three lines indicating a possible tendency toward differentiation. This study indicates that the endogenous p53 status of squamous cell carcinomas appears to play a critical role in determining the response to p53 adenoviral gene therapy. ^

PML tumor suppressor potentiates cell death through inhibition of the NF -kappa B survival pathway

The promyelocytic leukemia protein PML is a growth suppressor essential for induction of apoptosis by diverse apoptotic stimuli. The mechanism by which PML regulates cell death remains unclear. In this study we found that ectopic expression of PML potentiates cell death in the TNFα-resistant tumor line U2OS and significantly sensitized these cells to apoptosis induced by TNFα in a p53-independent manner. Our study demonstrated that both PML and PML/TNFα-induced cell death are associated with DNA fragmentation, activation of caspase-3, -7, -8, and degradation of DFF/ICAD. Furthermore, we found that PML-induced and PML/TNFα-induced cell death could be blocked by the caspase-8 inhibitors crmA and c-FLIP, but not by Bcl-2, the inhibitor of mitochondria-mediated apoptotic pathway. These findings indicate that this cell death event is initiated through the death receptor-dependent apoptosis pathway. Our study further showed that PML recruits NF-kappa B (NF-κB) to the PML nuclear body, blocks NF-κB binding to its cognate enhancer, and represses its transactivation function with the C-terminal region. Therefore PML inhibits the NF-κB survival pathway. Overexpression of NF-κB rescued cell death induced by PML and PML/TNFκ. These results imply that PML is a functional repressor of NF-κB. This notion was further supported by the finding that the PML−/− mouse embryo fibroblasts (MEFs) are more resistant than the wild-type MEFs to TNFκ-induced apoptosis. In conclusion, our studies convincingly demonstrated that PML potentiates cell death through inhibition of the NF-κB survival pathway. Activation of NF-κB frequently occurs during oncogenesis. Our study here suggests that a loss of PML function enhances the NF-κB survival pathway and this event may contribute to tumorigenesis. ^