Autophagy supports generation of cells with high CD44 expression via modulation of oxidative stress and Parkin-mediated mitochondrial clearance.

High CD44 expression is associated with enhanced malignant potential in esophageal squamous cell carcinoma (ESCC), among the deadliest of all human carcinomas. Although alterations in autophagy and CD44 expression are associated with poor patient outcomes in various cancer types, the relationship between autophagy and cells with high CD44 expression remains incompletely understood. In transformed oesophageal keratinocytes, CD44Low-CD24High (CD44L) cells give rise to CD44High-CD24-/Low (CD44H) cells via epithelial-mesenchymal transition (EMT) in response to transforming growth factor (TGF)-ß. We couple patient samples and xenotransplantation studies with this tractable in vitro system of CD44L to CD44H cell conversion to investigate the functional role of autophagy in generation of cells with high CD44 expression. We report that high expression of the autophagy marker cleaved LC3 expression correlates with poor clinical outcome in ESCC. In ESCC xenograft tumours, pharmacological autophagy inhibition with chloroquine derivatives depletes cells with high CD44 expression while promoting oxidative stress. Autophagic flux impairment during EMT-mediated CD44L to CD44H cell conversion in vitro induces mitochondrial dysfunction, oxidative stress and cell death. During CD44H cell generation, transformed keratinocytes display evidence of mitophagy, including mitochondrial fragmentation, decreased mitochondrial content and mitochondrial translocation of Parkin, essential in mitophagy. RNA interference-mediated Parkin depletion attenuates CD44H cell generation. These data suggest that autophagy facilitates EMT-mediated CD44H generation via modulation of redox homeostasis and Parkin-dependent mitochondrial clearance. This is the first report to implicate mitophagy in regulation of tumour cells with high CD44 expression, representing a potential novel therapeutic avenue in cancers where EMT and CD44H cells have been implicated, including ESCC.

Cellular senescence checkpoint function determines differential Notch1-dependent oncogenic and tumor-suppressor activities.

Notch activity regulates tumor biology in a context-dependent and complex manner. Notch may act as an oncogene or a tumor-suppressor gene even within the same tumor type. Recently, Notch signaling has been implicated in cellular senescence. Yet, it remains unclear as to how cellular senescence checkpoint functions may interact with Notch-mediated oncogenic and tumor-suppressor activities. Herein, we used genetically engineered human esophageal keratinocytes and esophageal squamous cell carcinoma cells to delineate the functional consequences of Notch activation and inhibition along with pharmacological intervention and RNA interference experiments. When expressed in a tetracycline-inducible manner, the ectopically expressed activated form of Notch1 (ICN1) displayed oncogene-like characteristics inducing cellular senescence corroborated by the induction of G0/G1 cell-cycle arrest, Rb dephosphorylation, flat and enlarged cell morphology and senescence-associated ß-galactosidase activity. Notch-induced senescence involves canonical CSL/RBPJ-dependent transcriptional activity and the p16(INK4A)-Rb pathway. Loss of p16(INK4A) or the presence of human papilloma virus (HPV) E6/E7 oncogene products not only prevented ICN1 from inducing senescence but permitted ICN1 to facilitate anchorage-independent colony formation and xenograft tumor growth with increased cell proliferation and reduced squamous-cell differentiation. Moreover, Notch1 appears to mediate replicative senescence as well as transforming growth factor-ß-induced cellular senescence in non-transformed cells and that HPV E6/E7 targets Notch1 for inactivation to prevent senescence, revealing a tumor-suppressor attribute of endogenous Notch1. In aggregate, cellular senescence checkpoint functions may influence dichotomous Notch activities in the neoplastic context.

A requirement for Nedd9 in luminal progenitor cells prior to mammary tumorigenesis in MMTV-HER2/ErbB2 mice.

Overexpression of the NEDD9/HEF1/Cas-L scaffolding protein is frequent, and drives invasion and metastasis in breast, head and neck, colorectal, melanoma, lung and other types of cancer. We have examined the consequences of genetic ablation of Nedd9 in the MMTV-HER2/ERBB2/neu mouse mammary tumor model. Unexpectedly, we found that only a limited effect on metastasis in MMTV-neu;Nedd9(-/-) mice compared with MMTV-neu;Nedd9(+/+) mice, but instead a dramatic reduction in tumor incidence (18 versus 80%), and a significantly increased latency until tumor appearance. Orthotopic reinjection and tail-vein injection of cells arising from tumors, coupled with in vivo analysis, indicated tumors arising in MMTV-neu;Nedd9(-/-) mice had undergone mutational selection that overcame the initial requirement for Nedd9. To better understand the defects in early tumor growth, we compared mammary progenitor cell pools from MMTV-neu;Nedd9(-/-) versus MMTV-neu;Nedd9(+/+) mice. The MMTV-neu;Nedd9(-/-) genotype selectively reduced both the number and colony-forming potential of mammary luminal epithelial progenitor cells, while not affecting basal epithelial progenitors. MMTV-neu;Nedd9(-/-) mammospheres had striking defects in morphology and cell polarity. All of these defects were seen predominantly in the context of the HER2/neu oncogene, and were not associated with randomization of the plane of mitotic division, but rather with depressed expression the cell attachment protein FAK, accompanied by increased sensitivity to small molecule inhibitors of FAK and SRC. Surprisingly, in spite of these significant differences, only minimal changes were observed in the gene expression profile of Nedd9(-/-) mice, indicating critical Nedd9-dependent differences in cell growth properties were mediated via post-transcriptional regulation of cell signaling. Coupled with emerging data indicating a role for NEDD9 in progenitor cell populations during the morphogenesis of other tissues, these results indicate a functional requirement for NEDD9 in the growth of mammary cancer progenitor cells.

Aurora kinase A mediates epithelial ovarian cancer cell migration and adhesion.

Aurora kinase A (AURKA) localizes to centrosomes and mitotic spindles where it mediates mitotic progression and chromosomal stability. Overexpression of AURKA is common in cancer, resulting in acquisition of alternate non-mitotic functions. In the current study, we identified a novel role for AURKA in regulating ovarian cancer cell dissemination and evaluated the efficacy of an AURKA-selective small molecule inhibitor, alisertib (MLN8237), as a single agent and combined with paclitaxel using an orthotopic xenograft model of epithelial ovarian cancer (EOC). Ovarian carcinoma cell lines were used to evaluate the effects of AURKA inhibition and overexpression on migration and adhesion. Pharmacological or RNA interference-mediated inhibition of AURKA significantly reduced ovarian carcinoma cell migration and adhesion and the activation-associated phosphorylation of the cytoskeletal regulatory protein SRC at tyrosine 416 (pSRC(Y416)). Conversely, enforced expression of AURKA resulted in increased migration, adhesion and activation of SRC in cultured cells. In vivo tumor growth and dissemination were inhibited by alisertib treatment as a single agent. Moreover, combination of alisertib with paclitaxel, an agent commonly used in treatment of EOC, resulted in more potent inhibition of tumor growth and dissemination compared with either drug alone. Taken together, these findings support a role for AURKA in EOC dissemination by regulating migration and adhesion. They also point to the potential utility of combining AURKA inhibitors with taxanes as a therapeutic strategy for the treatment of EOC patients.

Periostin cooperates with mutant p53 to mediate invasion through the induction of STAT1 signaling in the esophageal tumor microenvironment.

Periostin (POSTN), a matricellular protein, has been reported to be important in supporting tumor cell dissemination. However, the molecular mechanisms underlying POSTN function within the tumor microenvironment are poorly understood. In this study, we observe that the inducible knockdown of POSTN decreases esophageal squamous cell carcinoma (ESCC) tumor growth in vivo and demonstrate that POSTN cooperates with a conformational missense p53 mutation to enhance invasion. Pathway analyses reveal that invasive esophageal cells expressing POSTN and p53(R175H) mutation display activation of signal transducer and activator of transcription 1 (STAT1) target genes, suggesting that the induction of STAT1 and STAT1-related genes could foster a permissive microenvironment that facilitates invasion of esophageal epithelial cells into the extracellular matrix. Genetic knockdown of STAT1 in transformed esophageal epithelial cells underscores the importance of STAT1 in promoting invasion. Furthermore, we find that STAT1 is activated in ESCC xenograft tumors, but this activation is attenuated with inducible knockdown of POSTN in ESCC tumors. Overall, these results highlight the novel molecular mechanisms supporting the capacity of POSTN in mediating tumor invasion during ESCC development and have implications of therapeutic strategies targeting the tumor microenvironment.

Retinoblastoma tumor-suppressor protein phosphorylation and inactivation depend on direct interaction with Pin1.

Inactivation of the retinoblastoma protein (pRb) by phosphorylation triggers uncontrolled cell proliferation. Accordingly, activation of cyclin-dependent kinase (CDK)/cyclin complexes or downregulation of CDK inhibitors appears as a common event in human cancer. Here we show that Pin1 (protein interacting with NIMA (never in mitosis A)-1), a peptidylprolyl isomerase involved in the control of protein phosphorylation, is an essential mediator for inactivation of the pRb. Our results indicate that Pin1 controls cell proliferation by altering pRb phosphorylation without affecting CDK and protein phosphatase 1 and 2 activity. We demonstrated that Pin1 regulates tumor cell proliferation through direct interaction with the spacer domain of the pRb protein, and allows the interaction between CDK/cyclin complexes and pRb in mid/late G1. Phosphorylation of pRb Ser 608/612 is the crucial motif for Pin1 binding. We propose that Pin1 selectively boosts the switch from hypo- to hyper-phosphorylation of pRb in tumor cells. In addition, we demonstrate that the CDK pathway is responsible for the interaction of Pin1 and pRb. Prospectively, our findings therefore suggest that the synergism among CDK and Pin1 inhibitors holds great promise for targeted pharmacological treatment of cancer patients, with the possibility of reaching high effectiveness at tolerated doses.

The role of the Birt-Hogg-Dubé protein in mTOR activation and renal tumorigenesis.

Birt-Hogg-Dubé (BHD) syndrome is a tumor-suppressor gene disorder characterized by skin tumors, cystic lung disease and renal cell carcinoma. Very little is known about the molecular pathogenesis of BHD. Clinical similarities between BHD and tuberous sclerosis complex (TSC) suggest that the BHD and TSC proteins may function within a common pathway. The TSC proteins inhibit the activity of the mammalian target of rapamycin complex 1 (TORC1), and in Schizosaccharomyces pombe, Bhd and Tsc1/Tsc2 have opposing roles in the regulation of amino-acid homeostasis. We report here that in mammalian cells, downregulation of BHD reduces the phosphorylation of ribosomal protein S6, an indicator of TORC1 activity. To determine whether folliculin, the product of the BHD gene, regulates mammalian target of rapamycin activity in vivo, we generated a mouse with targeted inactivation of the Bhd gene. The mice developed spontaneous oncocytic cysts and tumors composed of cells that resemble the renal cell carcinomas in BHD patients. The cysts and tumors had low levels of phospho-S6. Taken together, these data indicate that folliculin regulates the activity of TORC1, and suggest a new paradigm in which both inappropriately high and inappropriately low levels of TORC1 activity can be associated with renal tumorigenesis.

Focused antibody response in plasma cell-infiltrated non-medullary (NOS) breast cancers.

Breast tumors with prominent plasma cell (PC) infiltrates often have a more favorable natural course that may plausibly be mediated by anti-tumor activity of the elicited antibodies. These breast tumor-associated PCs are typically IgG dominant in contrast to normal breast PCs, which are mainly IgA. It is our hypothesis that this PC infiltration represents a host immune response that is driven by one or more tumor antigens. Previously, we and others showed that medullary carcinoma (MC) had a focused repertoire and features suggestive of a protein antigen driven response. Infrequently, non-MC, not otherwise specified (NOS) breast tumors may exhibit heavy PC infiltrations, also of IgG isotype. In this first characterization of this favorable prognosis NOS subgroup, IgG heavy chain (Hc) and light chain (Lc) variable (V) regions from three PC-infiltrated NOS tumors were randomly cloned and sequenced. We found biased (V) gene usage by the infiltrating PCs and somatic hypermutation in the rearranged Ig Hc and Lc V regions that were compatible with antigenic selection of the progenitor B cells. The antibody response of NOS infiltrated breast cancer is repertoire-focused, with 13-68% of isolates being clonally reiterated in the samples. Each NOS patient used distinct Hc V-D-J and Lc V-J rearrangements, with her own immune response "footprint," but the overall pattern of gene usage followed that typical of exogenous antigen-induced immune responses. The data are consistent with the hypothesis that PC infiltrates infrequently arising in NOS tumors, as previously inferred for MC, are in response to one or more breast cancer-associated protein tumor antigens.

AKT induces senescence in primary esophageal epithelial cells but is permissive for differentiation as revealed in organotypic culture.

Epidermal growth factor receptor (EGFR) overexpression and activation is critical in the initiation and progression of cancers, especially those of epithelial origin. EGFR activation is associated with the induction of divergent signal transduction pathways and a gamut of cellular processes; however, the cell-type and tissue-type specificity conferred by certain pathways remains to be elucidated. In the context of the esophageal epithelium, a prototype stratified squamous epithelium, EGFR overexpression is relevant in the earliest events of carcinogenesis as modeled in a three-dimensional organotypic culture system. We demonstrate that the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway, and not the MEK/MAPK (mitogen-activated protein kinase) pathway, is preferentially activated in EGFR-mediated esophageal epithelial hyperplasia, a premalignant lesion. The hyperplasia was abolished with direct inhibition of PI3K and of AKT but not with inhibition of the MAPK pathway. With the introduction of an inducible AKT vector in both primary and immortalized esophageal epithelial cells, we find that AKT overexpression and activation is permissive for complete epithelial formation in organotypic culture, but imposes a growth constraint in cells grown in monolayer. In organotypic culture, AKT mediates changes related to cell shape and size with an expansion of the differentiated compartment.

Carcinogen-induced animal models of head and neck squamous cell carcinoma.

This unit provides two models of oral carcinogenesis (hamster cheek pouch and rat tongue), as well as more simple procedures to induce squamous cell carcinomas (SCCs) in mouse skin. The most significant usage of these models has been their application in studying the molecular pathology of the genesis and multi-step progression of tobacco-associated SCC. Nevertheless, these protocols have been used frequently as in vivo bioassays to demonstrate the chemopreventive and, to a lesser extent, the chemotherapeutic effects of numerous compounds. Several examples, using four different carcinogenesis protocols, are presented with detailed steps on how to elicit squamous lesions and when and how to apply test compounds that could have either cancer chemopreventive or chemotherapeutic effects.

Pleiotrophin expression correlates with melanocytic tumor progression and metastatic potential.

BACKGROUND: Gene expression profiling of melanoma and nevic tissue has demonstrated that pleiotrophin (PTN) is significantly overexpressed in human melanomas. METHODS: To further evaluate PTN expression in melanocytic lesions, protein immunohistochemistry was performed on the spectrum of melanocytic lesions. RESULTS: Melanocytic nevi were consistently negative (n=58). In contrast, the great majority of metastatic melanomas were positive (33/34, 97%). The analysis of 34 primary melanomas demonstrated PTN positivity in 20 lesions while 14 lesions were negative. Within the primary melanomas, PTN immunoreactivity was associated with metastasis (p=0.0004) and decreased melanoma-related survival (p=0.0444). Univariate analysis of PTN immunoreactivity predicted an increased risk for metastasis (relative risk 9.1, p=0.003). CONCLUSIONS: The results of this study confirm previous gene profiling data showing differential PTN expression between melanocytic nevi and melanomas. In addition, lesional PTN expression is associated with metastatic potential and may be a prognostic factor for melanomas.

Furin expression in squamous cell carcinomas of the oral cavity and other sites evaluated by tissue microarray technology.

Furin is a proprotein convertase that activates many cancer development-related substrates such as growth factors, growth factor-receptors, adhesion molecules, and matrix degrading enzymes. Furin expression was studied in sections from tissue microarrays (TMA) and conventional paraffin blocks in a collection of squamous cell carcinomas (SCC) from three different sites. A total of 118 SCCs from the oral cavity, lung and esophagus as well as 34 precursor lesions (intraepithelial neoplasia) from the oral and bronchial mucosae were studied by immunohistochemistry. Furin expression was notably higher in most precursor lesions than in normal epithelia. Tumors from either the TMAs or the conventional blocks showed significant differences when compared to the mostly negative normal epithelia. High levels of furin expression were observed in approximately 50% SCCs of three different sites as well as in precursor lesions of the oral and bronchial mucosae. In addition another 30% showed low furin expression that was localized in all tumor cells including those in a basaloid position. Normal epithelia sometimes showed low level expression but the normal basal cells were always negative. These results show that furin is up-regulated in SCCs from three different organs and validates its use as a tumor marker in both invasive and pre-invasive neoplasia.

Furin expression in squamous cell carcinomas of the oral cavity and other sites evaluated by tissue microarray technology

Furin is a proprotein convertase that activates many cancer development-related substrates such as growth factors, growth factor-receptors, adhesion molecules, and matrix degrading enzymes. Furin expression was studied in sections from tissue microarrays (TMA) and conventional paraffin blocks in a collection of squamous cell carcinomas (SCC) from three different sites. A total of 118 SCCs from the oral cavity, lung and esophagus as well as 34 precursor lesions (intraepithelial neoplasia) from the oral and bronchial mucosae were studied by immunohistochemistry. Furin expression was notably higher in most precursor lesions than in normal epithelia. Tumors from either the TMAs or the conventional blocks showed significant differences when compared to the mostly negative normal epithelia. High levels of furin expression were observed in approximately 50

SCCs of three different sites as well as in precursor lesions of the oral and bronchial mucosae. In addition another 30

showed low furin expression that was localized in all tumor cells including those in a basaloid position. Normal epithelia sometimes showed low level expression but the normal basal cells were always negative. These results show that furin is up-regulated in SCCs from three different organs and validates its use as a tumor marker in both invasive and pre-invasive neoplasia.

Furin expression in squamous cell carcinomas of the oral cavity and other sites evaluated by tissue microarray technology.

Furin is a proprotein convertase that activates many cancer development-related substrates such as growth factors, growth factor-receptors, adhesion molecules, and matrix degrading enzymes. Furin expression was studied in sections from tissue microarrays (TMA) and conventional paraffin blocks in a collection of squamous cell carcinomas (SCC) from three different sites. A total of 118 SCCs from the oral cavity, lung and esophagus as well as 34 precursor lesions (intraepithelial neoplasia) from the oral and bronchial mucosae were studied by immunohistochemistry. Furin expression was notably higher in most precursor lesions than in normal epithelia. Tumors from either the TMAs or the conventional blocks showed significant differences when compared to the mostly negative normal epithelia. High levels of furin expression were observed in approximately 50

SCCs of three different sites as well as in precursor lesions of the oral and bronchial mucosae. In addition another 30

showed low furin expression that was localized in all tumor cells including those in a basaloid position. Normal epithelia sometimes showed low level expression but the normal basal cells were always negative. These results show that furin is up-regulated in SCCs from three different organs and validates its use as a tumor marker in both invasive and pre-invasive neoplasia.

Elevated furin expression in aggressive human head and neck tumors and tumor cell lines.

Pro-protein convertases (PCs) are proteases that recognize and cleave precursor proteins. Furin, a well-studied PC, is ubiquitously expressed, and it has been implicated in many physiological and pathological processes. Some substrates for furin, such as membrane type 1 (MT1) matrix metalloproteinase (MMP), an MMP that activates gelatinase, a collagen-degrading enzyme, are associated with the advanced malignant phenotype. This report examines the expression of furin in carcinoma cell lines of different invasive ability. The levels of furin mRNA and protein correlated with the aggressiveness of tumor cell lines derived from head and neck and lung cancers. Furin expression also was investigated in primary head and neck squamous cell carcinomas (HNSCCs). Furin mRNA was not detected in nonmetastasizing carcinomas. In contrast, furin mRNA was expressed in metastasizing HNSCCs. Immunohistochemistry and Western blot analysis confirmed these results at the protein level. Furin activity was investigated indirectly by evaluating the expression of the pro-form and the processed form of MT1-MMP. Metastasizing HNSCCs showed increased expression of MT1-MMP. Furthermore, pro-MT1-MMP expression was noted in most of the nonmetastasizing HNSCCs analyzed by Western blot, and it was absent in the metastasizing HNSCCs. This finding suggests a lower level of furin-mediated MT1-MMP activation in the less aggressive cancers. These observations indicate that furin plays a role in tumor progression. Its overexpression in more aggressive or metastasizing cancers resulted in increased MMP processing.

Furin inhibition results in absent or decreased invasiveness and tumorigenicity of human cancer cells.

Pro-protein convertases such as furin are expressed in many human tumor lines and primary tumors. Furin processes stromelysin-3, membrane type 1 matrix metalloproteinase (MMPs) involved in tumor cell invasiveness, as well as growth factors such as transforming growth factor beta1. Evaluation of furin expression in head and neck squamous cell carcinoma (HNSCC) cells exhibiting different invasive ability showed that furin overexpression correlated with their respective invasiveness. The use of a selective furin inhibitor, alpha 1-PDX (PDX) was studied in three furin-expressing invasive HNSCC cell lines. The effects of PDX transfection were evaluated in vivo and in vitro to determine changes in the malignant phenotype. Transfection of HNSCC cell lines with PDX resulted in significant decrease or absence of tumorigenicity after s.c. inoculation into severe combined immunodeficient mice. Likewise, in vitro invasiveness was reduced approximately 50%. The in vivo invasion assay using tracheal xenotransplants showed even more drastic reductions of the invasive ability of PDX-transfected cells (up to an 80% decrease). PDX-transfected cells did not invade or penetrated less into the tracheal wall tissues than their vector alone-transfected counterparts. In addition, the former cells showed a remarkable decrease in MMP-2 processing and activity. After PDX transfection the cells were less efficient in processing the tumor progression-associated furin substrates transforming growth factor beta1 and pro-membrane type 1-MMP. These findings indicate that furin inhibition is a feasible approach to attenuate and even abolish certain critical attributes of the advanced malignant phenotype. Thus, furin should be considered as a promising target for cancer therapy.

Onset of natural killer cell lymphomas in transgenic mice carrying a truncated HMGI-C gene by the chronic stimulation of the IL-2 and IL-15 pathway.

Rearrangements of the high mobility group protein I-C (HMGI-C) gene, consisting in the loss of the carboxyl-terminal tail, have been frequently detected in benign human tumors of mesenchymal origin. We have previously demonstrated that transgenic (TG) mice carrying a truncated HMGI-C construct (HMGI-C/T) exhibit a giant phenotype together with a predominantly abdominal/pelvic lipomatosis. Here, we report that HMGI-C/T TG mice develop natural killer (NK)-T/NK cell lymphomas starting from 12 months of age. We found an increased expression of IL-2 and IL-15 proteins and their receptors in these lymphomas, and we demonstrate that HMGI-C/T protein positively regulates their expression in vitro. Therefore, the HMGI-C/T-mediated chronic stimulation of the IL-2/IL-15 pathway could be responsible for the onset of NK-T/NK cell lymphomas in HMGI-C/T TG mice.

Image cytometry of cyclin D1: a prognostic marker for head and neck squamous cell carcinomas.

CCND1 gene amplification and cyclin D1 protein overexpression are indicators for poor prognosis in invasive head and neck carcinomas. Increased CCND1 gene dosage is a more sensitive prognostic factor than protein overexpression as evaluated by conventional immunohistochemical techniques. Qualitative immunohistochemistry cannot distinguish cyclin D1 overexpression accompanied by amplification of the CCND1 gene from overexpression associated with normal CCND1 gene copy number. To improve the sensitivity of cyclin D1 protein determination, we applied quantitative techniques of image analysis to evaluate cyclin D1 in 54 head and neck carcinomas. There was a significantly higher rate of occurrence of adverse events (P = 0.043) among patients with CCND1 gene amplification than among those without gene amplification. There was a strong association between CCND1 gene amplification (as detected by Southern blot analysis) and the highest nuclear score (by image cytometry of the immunostained tumor sections). The predominance of cells in the lowest nuclear score category was significantly associated with normal copy number (P = 0.005). Conversely, the highest nuclear score was a significant predictor of gene dosage (P = 0.02). Similarly, high nuclear score was a good predictor of death as the final outcome of the disease (P = 0.01). Although somewhat less accurate than Southern blotting, image cytometry of immunohistochemical cyclin D1 stain appears to be a promising tool that could be useful for other tumor marker expression studies.

Exisulind, a novel proapoptotic drug, inhibits rat urinary bladder tumorigenesis.

Exisulind (Aptosyn) is a novel antineoplastic drug being developed for the prevention and treatment of precancerous and malignant diseases. In colon tumor cells, the drug induces apoptosis by a mechanism involving cyclic GMP (cGMP) phosphodiesterase inhibition, sustained elevation of cGMP, and protein kinase G activation. We studied the effect of exisulind on bladder tumorigenesis induced in rats by the carcinogen, N-butyl-N-(4-hydroxybutyl) nitrosamine. Exisulind at doses of 800, 1000, and 1200 mg/kg (diet) inhibited tumor multiplicity by 36, 47, and 64% and tumor incidence by 31, 38, and 61%, respectively. Experiments on the human bladder tumor cell line, HT1376, showed that exisulind inhibited growth with a GI(50) of 118 microM, suggesting that the antineoplastic activity of the drug in vivo involved a direct effect on neoplastic urothelium. Exisulind also induced apoptosis as determined by DNA fragmentation, caspase activation, and morphology. Analysis of phosphodiesterase (PDE) isozymes in HT1376 cells showed PDE5 and PDE4 isozymes that were inhibited by exisulind with IC(50)s of 112 and 116 microM, respectively. Inhibition of PDE5 appears to be pharmacologically relevant, because treatment of HT1376 cells increased cGMP and activated protein kinase G at doses that induce apoptosis, whereas cyclic AMP levels were not changed. Immunocytochemistry showed that PDE5 was localized in discrete perinuclear foci in HT1376 cells. Immunohistochemistry showed that PDE5 was overexpressed in human squamous and transitional cell carcinomas compared with normal urothelium. The data lead us to conclude that future clinical trials of exisulind for human bladder cancer treatment and/or prevention should be considered and suggest a mechanism of action involving cGMP-mediated apoptosis induction.

Mitochondria-to-nucleus stress signaling induces phenotypic changes, tumor progression and cell invasion.

Recently we showed that partial depletion of mitochondrial DNA (genetic stress) or treatment with mitochondrial-specific inhibitors (metabolic stress) induced a stress signaling that was associated with increased cytoplasmic-free Ca(2+) [Ca(2+)](c). In the present study we show that the mitochondria-to-nucleus stress signaling induces invasive phenotypes in otherwise non-invasive C2C12 myoblasts and human pulmonary carcinoma A549 cells. Tumor-specific markers cathepsin L and transforming growth factor beta (TGFbeta) are overexpressed in cells subjected to mitochondrial genetic as well as metabolic stress. C2C12 myoblasts subjected to stress showed 4- to 6-fold higher invasion through reconstituted Matrigel membrane as well as rat tracheal xenotransplants in Scid mice. Activation of Ca(2+)-dependent protein kinase C (PKC) under both genetic and metabolic stress conditions was associated with increased cathepsin L gene expression, which contributes to increased invasive property of cells. Reverted cells with approximately 70% of control cell mtDNA exhibited marker mRNA contents, cell morphology and invasive property closer to control cells. These results provide insights into a new pathway by which mitochondrial DNA and membrane damage can contribute to tumor progression and metastasis.