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PDF - 692K, c-Met activity is required for cell migration and invasion in MM cells from Nf2 (+/-);p16/p19 (+/-) mice. A) MM cell lines 380 and 129 were grown to confluence and subjected to a wound healing assay in presence or absence of c-Met inhibitor SU11274 (photos shown taken at 0 h and 24 h). Immunoblot analysis for P-c-Met, c-Met, P-AKT, P-ERK and ERK1/2 levels was performed 24 h post-treatment with SU11274. B) Results of Matrigel invasion assays performed with MM cell lines 87, 129 and 380 from Nf2+/-;p16/p19+/- mice, and MM cell line 680 from WT mouse, in presence or absence of SU11274. Invading cells stained with crystal violet, 24 h after treatment with vehicle or SU11274.

Berberine (BBR), an alkaloid component isolated from Chinese medicinal herb Huang Lian, has aroused broad interests for its antitumor effect in recent years. The signal transducer and activator of transcription 3 (STAT3), plays critical roles in malignant transformation and progression and was found to be constitutively activated in a variety of human cancers. In this study, we show that BBR inhibited cell proliferation, induced apoptosis, and suppressed tumor spheroid formation of lung cancer cell lines. These effects were correlated with BBR-mediated suppression of both phosphorylated and total levels of STAT3 protein. Furthermore, BBR promoted STAT3 degradation by enhancing ubiquitination. Importantly, we demonstrated that BBR was able to inhibit doxorubicin (DOX)-mediated STAT3 activation and sensitize lung cancer cells to the cytotoxic effect of DOX treatment. Given that BBR is widely used in clinic with low toxicity, our results are potentially important for the development of a novel combinatorial therapy with BBR and DOX in the treatment of lung cancer.

Malignant mesothelioma (MM) is a highly invasive and chemoresistant malignancy induced by asbestos fibers. NK4, a hepatocyte growth factor antagonist and angiogenesis inhibitor, consists of the N-terminal hairpin domain and four kringle domains of the α-chain of hepatocyte growth factor. The therapeutic potential of NK4 has been demonstrated in a variety of tumor types. However, the mechanisms by which NK4 inhibits tumor growth have not been well delineated. In this study, it is shown that the NK4 adenovirus (Ad-NK4) potently inhibits cell viability, invasiveness and tumorigenicity of human MM cells. Significantly, this study demonstrates for the first time that Ad-NK4 inhibits cancer stem-like cell (CSC) properties as assessed by spheroid formation assay, side population analysis and flow cytometric sorting of CD24 cells. In addition to inhibiting phosphorylation of Met and AKT, Ad-NK4 markedly suppressed the active form of β-catenin, a key mediator of both Wnt and AKT pathways. It is further demonstrated that expression of NK4 suppresses β-catenin nuclear localization and transcriptional activity. Intriguingly, the expression levels of Oct4 and Myc, two critical stem cell factors and downstream targets of β-catenin, were also diminished by Ad-NK4. Furthermore, the strong antitumor effect of NK4 was found to be linked to its ability to inhibit CSCs as revealed by immunohistochemical examination of tumor specimens from a mouse xenograft model of human MM. These findings suggest that NK4 acts as a CSC inhibitor by impeding Met/AKT/β-catenin signaling and holds promise for achieving durable therapeutic responses in MM by constraining the CSC component of these aggressive tumors.

Malignant mesothelioma is a highly aggressive, asbestos-related cancer frequently marked by mutations of both NF2 and CDKN2A. We demonstrate that germline knockout of one allele of each of these genes causes accelerated onset and progression of asbestos-induced malignant mesothelioma compared with asbestos-exposed Nf2+/− or wild-type mice. Ascites from some Nf2+/−;Cdkn2a+/− mice exhibited large tumor spheroids, and tail vein injections of malignant mesothelioma cells established from these mice, but not from Nf2+/− or wild-type mice, produced numerous tumors in the lung, suggesting increased metastatic potential of tumor cells from Nf2+/−;Cdkn2a+/− mice. Intraperitoneal injections of malignant mesothelioma cells derived from Nf2+/−;Cdkn2a+/− mice into severe combined immunodeficient mice produced tumors that penetrated the diaphragm and pleural cavity and harbored increased cancer stem cells (CSC). Malignant mesothelioma cells from Nf2+/−;Cdkn2a+/− mice stained positively for CSC markers and formed CSC spheroids in vitro more efficiently than counterparts from wild-type mice. Moreover, tumor cells from Nf2+/−;Cdkn2a+/− mice showed elevated c-Met expression/activation, which was partly dependent on p53-mediated regulation of miR-34a and required for tumor migration/invasiveness and maintenance of the CSC population. Collectively, these studies demonstrate in vivo that inactivation of Nf2 and Cdkn2a cooperate to drive the development of highly aggressive malignant mesotheliomas characterized by enhanced tumor spreading capability and the presence of a CSC population associated with p53/miR-34a–dependent activation of c-Met. These findings suggest that cooperativity between losses of Nf2 and Cdkn2a plays a fundamental role in driving the highly aggressive tumorigenic phenotype considered to be a hallmark of malignant mesothelioma. Cancer Res; 74(4); 1261–71. ©2013 AACR.

We report that the epidermal growth factor receptor (EGFR) pathway plays a critical role in regulating cancer stem-like cells (CSCs) in nasopharyngeal carcinoma (NPC), one of the most common malignant tumors in Southeast Asia. Effects of EGFR on maintaining CSCs are mainly mediated by AKT signaling, and β-catenin is responsible for governing CSC properties in response to EGFR/AKT activation. Significantly, CSCs are enriched by cisplatin and decreased by gefitinib in NPC xenograft models. Upon reimplantation in secondary mice, tumor cells derived from cisplatin-treated mice grew rapidly, whereas regrowth of tumor cells from gefitinib-treated mice was severely diminished. We further demonstrate that expression of EGFR correlates with expression of β-catenin and Nanog in primary tumor specimens from NPC patients. These findings provide mechanistic and preclinical evidence supporting the use of gefitinib alone or in combination with a chemotherapeutic agent in first-line therapy for patients with NPC. In addition, our results suggest that targeting β-catenin represents a rational clinical modality for patients whose tumors harbor activated EGFR or AKT.

// Linlin Li 1, 2, 3, * , Lingqun Zhu 1, * , Bingtao Hao 1, 2, 3 , Wenwen Gao 1 , Qianli Wang 1 , Keyi Li 1 , Meng Wang 1 , Mengqiu Huang 1 , Zhengjun Liu 4 , Qiaohong Yang 5, 6 , Xiqing Li 7 , Zhuo Zhong 8 , Wenhua Huang 9 , Guanghui Xiao 1 , Yang Xu 1, 2, 3 , Kaitai Yao 1 , Qiuzhen Liu 1, 2, 3 1 Cancer Research Institute, Southern Medical University, Guangzhou 510515, China 2 Guangdong Provincial Key Laboratory of Cancer Immunotherapy Research, Guangzhou 510515, China 3 Guangzhou Key Laboratory of Tumor Immunology Research, Southern Medical University, Guangzhou 510515, China 4 Department of Vascular Surgery, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China 5 School of Basic Medical Sciences, Guangzhou University of Chinese Medicine, Guangzhou 510006, China 6 Department of Pathology, Yale University School of Medicine, New Haven, CT 06520, USA 7 Department of Oncology, Henan Provincial People’s Hospital, Zhengzhou 450003, Henan, China 8 Department of Oncology, Guangzhou Hospital of Integrated Traditional and Western Medicine, Guangzhou 510800, China 9 Department of Human Anatomy, Southern Medical University, Guangzhou 510515, China * These authors have contributed equally to this work Correspondence to: Qiuzhen Liu, email: liuqiuzhen@126.com Kaitai Yao, email: yao.kaitai@hotmail.com Keywords: nitric oxide, iNOS, Warburg effect, PKM2, EGFR/ERK2 Received: October 19, 2016 Accepted: March 15, 2017 Published: March 23, 2017 ABSTRACT Aerobic glycolysis is essential for tumor growth and survival. Activation of multiple carcinogenic signals contributes to metabolism reprogramming during malignant transformation of cancer. Recently nitric oxide has been noted to promote glycolysis but the mechanism remains elusive. We report here the dual role of nitric oxide in glycolysis: low/physiological nitric oxide (≤ 100 nM) promotes glycolysis for ATP production, oxidative defense and cell proliferation of ovary cancer cells, whereas excess nitric oxide (≥ 500 nM) inhibits it. Nitric oxide has a positive effect on glycolysis by inducing PKM2 nuclear translocation in an EGFR/ERK2 signaling-dependent manner. Moreover, iNOS induced by mild inflammatory stimulation increased glycolysis and cell proliferation by producing low doses of nitric oxide, while hyper inflammation induced iNOS inhibited it by producing excess nitric oxide. Finally, iNOS expression is abnormally increased in ovarian cancer tissues and is correlated with PKM2 expression. Overexpression of iNOS is associated with aggressive phenotype and poor survival outcome in ovarian cancer patients. Our study indicated that iNOS/NO play a dual role of in tumor glycolysis and progression, and established a bridge between iNOS/NO signaling pathway and EGFR/ERK2/PKM2 signaling pathway, suggesting that interfering glycolysis by targeting the iNOS/NO/PKM2 axis may be a valuable new therapeutic approach of treating ovarian cancer.

// Qian-Qian Li 1, * , You-Ke Xie 1, * , Yue Wu 1 , Lin-Lin Li 1 , Ying Liu 1 , Xiao-Bo Miao 1 , Qiu-Zhen Liu 1 , Kai-Tai Yao 1 , Guang-Hui Xiao 1 1 Cancer Institute, Southern Medical University, Guangzhou 510515, China * These authors have contributed equally to this work Correspondence to: Guang-Hui Xiao, email: ghxiaohh@hotmail.com Keywords: sulforaphane, c-MYC, miR-214, cisplatin, lung cancer Received: June 11, 2016 Accepted: December 27, 2016 Published: January 05, 2017 ABSTRACT We herein report that sulforaphane (SFN), a potent anti-cancer and well-tolerated dietary compound, inhibits cancer stem-like cell (CSC) properties and enhances therapeutic efficacy of cisplatin in human non-small cell lung cancer (NSCLC). SFN exerted these functions through upregulation of miR-214, which in turn targets the coding region of c-MYC . This finding was further corroborated by our observations that plasmid or lentiviral vector-mediated expression of 3'UTR-less c-MYC cDNA and cisplatin- or doxorubicin-induced endogenous c-MYC accumulation was similarly suppressed by either SFN or miR-214. Further, we showed that the reported inhibitory effects of SFN on β-catenin are also mediated by miR-214. SFN/miR-214 signaling inhibited CSC properties and enhanced the cytotoxicity of chemotherapeutic drugs in vitro . Experiments with nude mice carrying xenograft tumors showed that SFN sensitized NSCLC cells to cisplatin's efficacy, which is accompanied by inhibition of cisplatin-induced c-MYC accumulation in tumor tissues. Our results provided strong evidence and mechanisms to support consideration of SFN or synthetic derivatives as a therapeutic agent in combination with cisplatin for the treatment of patients with NSCLC and, potentially, other types of c-MYC-addicted tumors.

The phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt) signaling pathway is one of the major oncogenic pathways and is activated in many types of human cancers, including hepatocellular carcinoma. It can also be activated by the hepatitis C virus (HCV) nonstructural 5A (NS5A) protein. In the present study, we set out to determine the regulatory effects of this pathway on the replication of hepatitis B virus (HBV). Our results demonstrate that the expression of a constitutively active Akt1 profoundly inhibited HBV RNA transcription and consequently reduced HBV DNA replication in HepG2 cells. This suppression of HBV gene transcription was apparently mediated by the activation of mTOR, as it was abolished by the mTOR inhibitor rapamycin. Moreover, treatment of HBV-expressing HepG2.2.15 cells with inhibitors of PI3K, Akt, and mTOR increased the transcription of 3.5-kb and 2.4-kb viral RNA as well as the replication of HBV DNA. This observation implies that the basal level activation of this pathway in HepG2 cells regulated HBV replication. Consistent with previous reports showing that the HCV NS5A protein could bind to the p85 subunit of PI3K and activate the PI3K-Akt signal transduction pathway, our results showed that expression of this protein could inhibit HBV RNA transcription and reduce HBV DNA replication in HepG2 cells. Taken together, our results suggest that the activation of the PI3K-Akt pathway during liver oncogenesis may be at least partially responsible for the elimination of HBV replication from tumor cells and may also provide an explanation for the observed suppression of HBV replication by HCV coinfection.

Production of β‐ketoacyl‐CoA, which is catalyzed by 3‐ketoacyl‐CoA synthase (KCS), is the first step in very long chain fatty acid (VLCFA) biosynthesis. Here we identified 58 KCS genes from Gossypium hirsutum, 31 from G. arboreum and 33 from G. raimondii by searching the assembled cotton genomes. The gene family was divided into the plant‐specific FAE1‐type and the more general ELO‐type. KCS transcripts were widely expressed and 32 of them showed distinct subgenome‐specific expressions in one or more cotton tissues/organs studied. Six GhKCS genes rescued the lethality of elo2Δelo3Δ yeast double mutant, indicating that this gene family possesses diversified functions. Most KCS genes with GA‐responsive elements (GAREs) in the promoters were significantly upregulated by gibberellin A3 (GA). Exogenous GA3 not only promoted fiber length, but also increased the thickness of cell walls significantly. GAREs present also in the promoters of several cellulose synthase (CesA) genes required for cell wall biosynthesis and they were all induced significantly by GA3. Because GA treatment resulted in longer cotton fibers with thicker cell walls and higher dry weight per unit cell length, we suggest that it may regulate fiber elongation upstream of the VLCFA‐ethylene pathway and also in the downstream steps towards cell wall synthesis.

The accessory human immunodeficiency virus type 1 (HIV-1) protein Nef activates the autophosphorylation activity of p21-activated kinase 2 (PAK2). Merlin, a cellular substrate of PAK2, is homologous to the ezrin-radixin-moesin family and plays a critical role in Rac signaling. To assess the possible impact on host cell metabolism of Nef-induced PAK2 activation, we investigated the phosphorylation of merlin in Nef expressing cells. Here we report that Nef induces merlin phosphorylation in multiple cell lines independently of protein kinase A. This intracellular phosphorylation of merlin directly correlates with in vitro assay of the autophosphorylation activity of Nef-activated PAK2. Importantly, merlin phosphorylation induced by Nef was also observed in human primary T cells. The finding that Nef induces phosphorylation of the key signaling molecule merlin suggests several possible roles for PAK2 activation in HIV pathogenesis.

Neurofibromatosis type II (NF2) is an autosomal dominant cancer syndrome characterized by the formation of tumors of the nervous system, particularly schwannomas and meningiomas. The NF2 gene is also implicated in the development of sporadic schwannomas and meningiomas, as well as tumor types seemingly unrelated to the NF2 disorder, such as malignant mesotheliomas. Inactivation of NF2 occurs by a "two-hit" mechanism, as proposed by Al Knudson, and the NF2 gene behaves as a classical tumor suppressor gene. The NF2 gene product, merlin, exhibits homology with the ezrin-radixin-moesin family of membrane-cytoskeleton-linking proteins. During the past several years, there has been intensive investigation aimed at elucidating the mechanisms underlying merlin's functions. In this review, we summarize the involvement of NF2 inactivation in tumorigenesis. We also discuss observations implicating merlin in cell motility and cell proliferation, with a focus on recent findings linking merlin to Rac signaling.

The FHIT gene has been implicated as a tumor suppressor gene in human malignancies. To determine if FHIT alterations play a role in human squamous cell carcinogenesis of the head and neck (HNSCC), we examined the gene and its product by RT-PCR, SSCP, Northern, Southern, and Western blot analysis in primary HNSCC and/or HNSCC cell lines. Three of 32 tumor samples lacked detectable expression of FHIT by RT-PCR but showed amplification of a control gene of similar size. One of 29 primary tumors and 2/9 HNSCC cell lines exhibited aberrant transcripts generated by RT-PCR methods using one set of 40 cycles of amplification. FHIT mRNA expression was absent in seven HNSCC cell lines but detectable in primary keratinocytes by Northern analysis. Using specific polyclonal antiserum to the full-length FHIT protein in immunoblot analyses, 4/9 cell lines analysed showed no expression of pFhit, two exhibited low levels of expression, and three expressed a putative truncated pFhit. One of 15 tumors analysed also exhibited an overexpressed truncated protein. PCR/SSCP and Southern analysis of one cell line DNA that expressed a truncated protein indicated that it sustained homozygous loss of FHIT exon 5. Our results suggest that alterations in FHIT at the DNA, RNA, and protein levels exist at a low but significant frequency in HNSCCs. Further studies regarding the potential biological activity of FHIT are needed to clarify the role of this gene in HNSCC tumorigenesis.

Background: The fragile histidine triad (FHIT) gene at chromosome 3p14.2 has been proposed to be a candidate tumor suppressor gene in human cancers. To test whether FHIT exhibits the functional properties of a tumor suppressor gene, we studied the expression of its protein (pFHIT) in human carcinoma cells and examined the ability of FHIT to inhibit the neoplastic phenotype of cancer cells. Methods: Subcellular localization and patterns of protein expression in tumor cells were determined by immunohistochemical analysis and immunoblotting with the use of polyclonal antipFHIT antisera. In tumor cells with undetectable pFHIT, we examined the effect of recombinant pFHIT expression on morphology, growth rate, colony formation, and in vivo tumor formation. Results: We demonstrated that pFHIT is a cytoplasmic 17-kd polypeptide whose expression could not be detected in 30 of 52 human carcinoma cell lines tested. We observed, however, that the stable overexpression of pFHIT did not alter cell morphology, inhibit colony formation, or inhibit cell proliferation in vitro. Furthermore, overexpression of pFHIT did not lead to altered cell cycle kinetics in dividing cells. The in vivo tumorigenicity of a tumor cell line that expressed high levels of recombinant pFHIT was equivalent to that of control transfectants and of parental cells. Conclusions: These results suggest that the replacement of pFHIT in human carcinoma cells does not suppress tumor cell growth and that this protein may be involved in tumorigenesis in ways that are distinct from the ‘‘classic’’ tumor suppressor paradigm. [J Natl Cancer Inst 1998;90: 426‐32]

Tuberous sclerosis complex (TSC) is characterized by the appearance of nonmalignant tumors that affect a wide spectrum of organs, including the heart. TSC disease-causing genes have been identified on chromosomes 9 (TSC1) and 16 (TSC2). This study examined the impact of the TSC2 gene product on cardiomyocyte proliferation and terminal differentiation. We took advantage of the observation that Eker rats carry a germ-line TSC2 mutation. Rats heterozygous for the mutation (TSC2EK/+) are predisposed to renal carcinoma, whereas animals homozygous for the mutation (TSC2EK/EK) die in utero during midgestation. Spontaneously contractile cardiomyocytes were observed after multiple passages of whole embryo cultures prepared from embryonic day 12.5 TSC2EK/EK fetuses but not from TSC2EK/+ or wild-type fetuses. The TSC2EK/EK cardiomyocytes continued to actively synthesize DNA after as many as eight passages. Cytological, ultrastructural, and molecular analyses indicated that the TSC2EK/EK cardiomyocytes retained a highly differentiated phenotype similar to that observed for normal rat cardiomyocytes during late embryonic and early neonatal life. These results suggested that the TSC2 gene product is required for normal cardiomyocyte cell-cycle withdrawal and terminal differentiation.

The Tsc2 gene, which is mutationally inactivated in the germ line of some families with tuberous sclerosis, encodes a large, membrane-associated GTPase activating protein (GAP) designated tuberin. Studies of the Eker rat model of hereditary cancer strongly support the role of Tsc2 as a tumor suppressor gene. In this study, the biological activity of tuberin was assessed by expressing the wild-type Tsc2 gene in tumor cell lines lacking functional tuberin and also in rat fibroblasts with normal levels of endogenous tuberin. The colony forming efficiency of Eker rat-derived renal carcinoma cells was significantly reduced following reintroduction of wild-type Tsc2. Tumor cells expressing the transfected Tsc2 gene became more anchorage-dependent and lost their ability to form tumors in severe combined immunodeficient mice. At the cellular level, restoration of tuberin expression caused morphological changes characterized by enlargement of the cells and increased contact inhibition. As with the full-length Tsc2 gene, a clone encoding only the C terminus of tuberin (amino acids 1049-1809, including the GAP domain) was capable of reducing both colony formation and in vivo tumorigenicity when transfected into the Eker rat tumor cells. In normal Rat1 fibroblasts, conditional overexpression of tuberin also suppressed colony formation and cell growth in vitro. These results provide direct experimental evidence for the tumor suppressor function of Tsc2 and suggest that the tuberin C terminus plays an important role in this activity.

Somatic events leading to the inactivation of tumor suppressor genes often involve chromosomal alterations that can be detected as loss of heterozygosity (LOH). In the Eker rat, spontaneous tumors of the kidney, uterus, and spleen develop as a result of a germline mutation of the tuberous sclerosis 2 (Tsc2) gene. We examined the pattern and frequency of LOH at the predisposing locus in 77 primary tumors and cell lines to gain an understanding of the role of Tsc2 allelic loss in the pathogenesis of Eker-derived tumors. Although most renal and uterine tumors (primary and cell lines) displayed LOH, splenic hemangiosarcomas did not. Although the presence of normal tissue may account for some of this difference, the possibility exists that an alternative mechanism, such as subtle mutation or gene dosage effects, may be involved during splenic tumorigenesis. Northern analysis confirmed that LOH resulted in loss of the wild-type transcripts for the Tsc2 gene. Thus, the inactivation of both alleles plays an important role in renal and uterine tumor development, in keeping with Knudson's two-hit hypothesis. In addition, renal tumors that retained the wild-type allele also did not express the normal transcript, suggesting that the remaining Tsc2 alleles had acquired subtle mutations resulting in loss of gene function.

The neurofibromatosis type 2 NF2 gene product, merlin, is a tumor suppressor frequently inactivated in malignant mesothelioma (MM). To investigate a possible correlation between merlin inactivation and MM invasiveness, we restored merlin expression in NF2-deficient MM cells. Re-expression of merlin markedly inhibited cell motility, spreading and invasiveness, properties connected with the malignant phenotype of MM cells. To test directly whether merlin inactivation promotes invasion in a nonmalignant system, we used small interfering RNA to silence Nf2 in mouse embryonic fibroblasts (MEFs) and found that downregulation of merlin resulted in enhanced cell spreading and invasion. To delineate signaling events connected with this phenotype, we investigated the effect of merlin expression on focal adhesion kinase (FAK), a key component of cellular pathways affecting migration and invasion. Expression of merlin attenuated FAK phosphorylation at the critical phosphorylation site Tyr397 and disrupted the interaction of FAK with its binding partners Src and p85, the regulatory subunit of phosphatidylinositol-3-kinase. In addition, NF2-null MM cells stably overexpressing FAK showed increased invasiveness, which decreased significantly when merlin expression was restored. Collectively, these findings suggest that merlin inactivation is a critical step in MM pathogenesis and is related, at least in part, with upregulation of FAK activity.

Malignant mesotheliomas (MMs) are very aggressive tumors that respond poorly to standard chemotherapeutic approaches. The phosphatidylinositol 3-kinase (PI3K)/AKT pathway has been implicated in tumor aggressiveness, in part by mediating cell survival and reducing sensitivity to chemotherapy. Using antibodies recognizing the phosphorylated/activated form of AKT kinases, we observed elevated phospho-AKT staining in 17 of 26 (65%) human MM specimens. In addition, AKT phosphorylation was consistently observed in MMs arising in asbestos-treated mice and in MM cell xenografts. Consistent with reports implicating hepatocyte growth factor (HGF)/Met receptor signaling in MM, all 14 human and murine MM cell lines had HGF-inducible AKT activity. One of nine human MM cell lines had elevated AKT activity under serum-starvation conditions, which was associated with a homozygous deletion of PTEN, the first reported in MM. Treatment of this cell line with the mTOR inhibitor rapamycin resulted in growth arrest in G1 phase. Treatment of MM cells with the PI3K inhibitor LY294002 in combination with cisplatin had greater efficacy in inhibiting cell proliferation and inducing apoptosis than either agent alone. Collectively, these data indicate that MMs frequently express elevated AKT activity, which may be targeted pharmacologically to enhance chemotherapeutic efficacy. These findings also suggest that mouse models of MM may be useful for future preclinical studies of pharmaceuticals targeting the PI3K/AKT pathway.

Inactivation of the NF2 tumor suppressor gene has been observed in certain benign and malignant tumors. Recent studies have demonstrated that merlin, the product of the NF2 gene, is regulated by Rac/PAK signaling. However, the mechanism by which merlin acts as a tumor suppressor has remained obscure. In this report, we show that adenovirus-mediated expression of merlin in NF2-deficient tumor cells inhibits cell proliferation and arrests cells at G1 phase, concomitant with decreased expression of cyclin D1, inhibition of CDK4 activity, and dephosphorylation of pRB. The effect of merlin on cell cycle progression was partially overridden by ectopic expression of cyclin D1. RNA interference experiments showed that silencing of the endogenous NF2 gene results in upregulation of cyclin D1 and S-phase entry. Furthermore, PAK1-stimulated cyclin D1 promoter activity was repressed by cotransfection of NF2, and PAK activity was inhibited by expression of merlin. Interestingly, the S518A mutant form of merlin, which is refractory to phosphorylation by PAK, was more efficient than the wild-type protein in inhibiting cell cycle progression and in repressing cyclin D1 promoter activity. Collectively, our data indicate that merlin exerts its antiproliferative effect, at least in part, via repression of PAK-induced cyclin D1 expression, suggesting a unifying mechanism by which merlin inactivation might contribute to the overgrowth seen in both noninvasive and malignant tumors.

Background Neurofibromatosis type 2 is a group of tumors caused by loss-of-function mutations of a tumor suppressor gene encoding NF2/merlin. Development of chemotherapeutics for this disease, which often threatens the life of young children, has been hampered by a limited information on the signaling function of NF2. NF2 can inhibit Ras-induced malignant transformation. However, the primary (signaling) target of NF2 in the oncogenic pathway has not been previously identified. Results Here, using a series of NF2 constructs, we show that NF2 inhibits directly the Rac/CDC42-dependent Ser/Thr kinase PAK1, which is essential for both Ras transformation and neurofibromatosis type 1 (NF1), through two separate domains. A mutant of NF2, that lacks the PAK1-inhibiting domain of 78 amino acids (NF78C, residues 447-524), fails to suppress Ras transformation. Furthermore, PAK1-specific inhibitors CEP-1347 and WR-PAK18 selectively inhibit the growth of NF2-deficient cancer cells, but not NF2-positive cells. Conclusions These results suggest that PAK1 is essential for the malignant growth of NF2-deficient cells, and that PAK1-blocking drugs could be potentially useful forthe treatment of neurofibromatosis types 2, in addition to Ras-induced cancers and neurofibromatosis type 1.

The neurofibromatosis type 2 tumor suppressor gene, NF2, is mutated in the germ line of NF2 patients and predisposes affected individuals to intracranial and spinal tumors. Moreover, somatic mutations of NF2 can occur in the sporadic counterparts of these neurological tumor types as well as in certain neoplasms of non-neuroectodermal origin, such as malignant mesothelioma and melanoma. NF2 encodes a 595-amino acid protein, merlin, which exhibits significant homology to the ezrin-radixin-moesin family of proteins. However, the mechanism by which merlin exerts its tumor suppressor activity is not well understood. In this investigation, we show that merlin is phosphorylated in response to expression of activated Rac and activated Cdc42 in mammalian cells. Furthermore, we demonstrate that merlin phosphorylation is mediated by p21-activated kinase (Pak), a common downstream target of both Rac and Cdc42. Both in vivo and in vitro kinase assays demonstrated that Pak can directly phosphorylate merlin at serine 518, a site that affects merlin activity and localization. These biochemical investigations provide insights into the regulation of merlin function and establish a framework for elucidating tumorigenic mechanisms involved in neoplasms associated with merlin inactivation.

Hepatocyte growth factor (HGF) is a ligand of the receptor tyrosine kinase encoded by the c- Met protooncogene. HGF/Met signaling has multifunctional effects on various cell types. We sought to determine the role of HGF/Met in apoptosis and identify signal transducers involved in this process. In experiments with human SK-LMS-1 leiomyosarcoma cells, we show that the Akt kinase is activated by HGF in a time- and dose-dependent manner by phosphatidylinositol 3-kinase (PI3-kinase). Akt is also activated by active tumorigenic forms of Met, i.e., ligand-independent Tpr-Met, a truncated and constitutively dimerized form of Met, and a mutationally activated version of Met corresponding to that found in human hereditary papillary renal carcinoma. In NIH 3T3 cells transfected with wild-type Met, HGF inhibits apoptosis induced by serum starvation and UV irradiation. HGF-induced survival correlates with Akt activity and is inhibited by the specific PI3-kinase inhibitor LY294002, indicating that HGF inhibits cell death through the PI3-kinase/Akt signal transduction pathway. Furthermore, transiently transfected Tpr-Met activates Akt (both Akt1 and Akt2) and protects cells from apoptosis. Mitogen-activated protein kinase (MAPK) also is activated by HGF and rescues cells from apoptosis, although the cytoprotective effect is less marked than for PI3-kinase/Akt. Blocking MAPK with the specific MAPK kinase inhibitor PD098059 impairs the ability of HGF to promote cell survival. Similar results were obtained with NIH 3T3 cells expressing the fusion protein Trk-Met and stimulated with nerve growth factor, the Trk ligand. These results demonstrate that HGF/Met is capable of protecting cells from apoptosis by using both PI3-kinase/Akt and, to a lesser extent, MAPK pathways.

The tuberous sclerosis complex 2 (TSC2) is a tumor suppressor gene that plays a causative role in the autosomal dominant syndrome of tuberous sclerosis. The latter is characterized by the development of hamartomas and occasional malignancies. Expression of the wild-type gene in TSC2 mutant tumor cells inhibits proliferation and tumorigenicity. This "suppressor" activity is encoded by functional domain(s) in the C terminus that contains homology to Rap1GAP. Using a yeast two-hybrid assay to identify proteins that interact with the C-terminal domain of tuberin, the product of TSC2, a cytosolic factor, rabaptin-5, was found to associate with a distinct domain lying adjacent to the TSC2 GAP homology region. Rabaptin-5 also binds the active form of GTPase Rab5. Immune complexes of native tuberin, as well as recombinant protein, possessed activity to stimulate GTP hydrolysis of Rab5. Tuberin GAP activity was specific for Rab5 and showed no cross-reactivity with Rab3a or Rab6. Cells lacking tuberin possessed minimal Rab5GAP activity and were associated with an increased uptake of horseradish peroxidase. Re-expression of tuberin in TSC2 mutant cells reduced the rate of fluid-phase endocytosis. These findings suggest that tuberin functions as a Rab5GAP in vivo to negatively regulate Rab5-GTP activity in endocytosis.

Genetic predisposition to neoplasia often involves tumor suppressor genes. One such model of hereditary renal carcinoma was described in the rat by Eker. These tumors share morphologic similarities with human renal cancer. Linkage analysis localized the inherited mutation to rat chromosome band 10q12. This region is syntenic with human chromosome band 16p13.3, the site of the tuberous sclerosis 2 (TSC2) gene. A specific rearrangement of the rat homologue of TSC2 was found to cosegregate with carriers of the predisposing mutation. Tumors with or without loss of heterozygosity expressed only the mutant allele, consistent with the two-hit hypothesis. This mutation gave rise to an aberrant transcript that deletes the 3' end normally containing a region of homology with the catalytic domain of rap1GAP.

Malignant mesothelioma (MM) is a highly invasive and chemoresistant malignancy induced by asbestos fibers. Hepatocyte growth factor (HGF) and its receptor Met play a critical role in the pathogenesis of MM, and targeting HGF/Met signaling could be therapeutically important. NK4, a HGF antagonist and angiogenesis inhibitor, consists of the N-terminal hairpin domain and four kringle domains of the -chain of HGF. The therapeutic potential of NK4 has been demonstrated in several types of tumors. However the mechanisms by which NK4 inhibits tumor growth have not been well elucidated. In this study, we show that the NK4 adenovirus potently inhibits cell viability, invasiveness, migration, and spreading of human MM cells in vitro. Significantly, we demonstrated for the first time that NK4 reduced expression levels of stem cell markers as assessed by western blot analysis and inhibited self-renewal of MM stem cells as measured by spheroid formation assay. Furthermore, NK4 adenovirus had strong antitumor and anti-angiogenesis effects in two different mouse models of MM. These findings suggest that NK4 holds promise as a therapeutic agent for patients with MM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A64.

Background Although the prognostic roles of β-catenin expression in non-small cell lung cancer (NSCLC) have been reported in several immunohistochemical (IHC) studies, the results were not consistent because some studies lack sufficient number of the positive cases or did not evaluate the subcellular localization features of the protein. Method In this study, we have evaluated the expression levels and subcellular localization of β-catenin and Nanog proteins IHC staining in tissue specimens from 309 patients with NSCLC, and explored their association with clinicopathological features and patient outcome. Results We showed that patients with negative expression of membranous beta-catenin had a trend towards shorter survival (p=0.064) than those with positive expression. In contrast to previous studies, we found that increased expression of either cytoplasmic or nuclear β-catenin was strongly associated with poor prognosis and was an independent prognosticator for overall survival (p p p Conclusion We propose that evaluation of subcellular localization of β-catenin and Nanog expression is of clinical significance for patients with NSCLC.