To investigate the role of the Fhit gene in carcinogen induction of neoplasia, we have inactivated one Fhit allele in mouse embryonic stem cells and produced (129͞SvJ ؋ C57BL͞6J) F1 mice with a Fhit allele inactivated (؉͞؊). Fhit ؉͞؉ and ؉͞؊ mice were treated intragastrically with nitrosomethylbenzylamine and observed for 10 wk posttreatment. A total of 25% of the ؉͞؉ mice developed adenoma or papilloma of the forestomach, whereas 100% of the ؉͞؊ mice developed multiple tumors that were a mixture of adenomas, squamous papillomas, invasive carcinomas of the forestomach, as well as tumors of sebaceous glands. The visceral and sebaceous tumors, which lacked Fhit protein, were similar to those characteristic of Muir-Torre familial cancer syndrome.esophageal͞gastric cancer ͉ N-nitrosomethylbenzylamine ͉ carcinogeninduced tumorigenesis ͉ Fhit knockout mice ͉ tumor suppressor gene

Mice carrying one inactivated Fhit allele (Fhit ؉͞؊ mice) are highly susceptible to tumor induction by N-nitrosomethylbenzylamine, with 100% of Fhit ؉͞؊ mice exhibiting tumors of the forestomach͞ squamocolumnar junction vs. 25% of Fhit ؉͞؉ controls. In the current study a single N-nitrosomethylbenzylamine dose was administered to Fhit ؉͞؉, ؉͞؊, and ؊͞؊ mice to compare carcinogen susceptibility in ؉͞-and ؊͞؊ Fhit-deficient mice. At 29 weeks after treatment, 7.7% of wild-type mice had tumors. Of the Fhit ؊͞؊ mice 89.5% exhibited tumors (average 3.3 tumors͞mouse) of the forestomach and squamocolumnar junction; half of the ؊͞؊ mice had medium (2 mm diameter) to large (>2 mm) tumors. Of the Fhit ؉͞؊ mice 78% exhibited tumors (average 2.4 tumors͞mouse) and 22% showed medium to large tumors. Untreated Fhit-deficient mice have been observed for up to 2 years for spontaneous tumors. Fhit ؉͞؊ mice (average age 21 mo) exhibit an average of 0.94 tumors of different types; Fhit ؊͞؊ mice (average age 16 mo) also showed an array of tumors (average 0.76 tumor͞mouse). The similar spontaneous and induced tumor spectra observed in mice with one or both Fhit alleles inactivated suggests that Fhit may be a one-hit tumor suppressor gene in some tissues. D eletion at chromosome region 3p14.2 is among the earliest events observed in many cancers and is observed in very early preneoplastic lesions of lung, cervix, and breast (1-5). The Ϸ2-Mb fragile FHIT gene is the likely target of these diseaseassociated deletions, which result in loss of portions of one or both FHIT alleles, with concomitant loss of at least half of the normal complement of Fhit protein. Whether loss of one FHIT allele predisposes to further genetic changes is not known, but clonal expansion of the 3p14.2-deleted cells occurs. In the majority of frankly malignant lesions of most cancer types, one or both FHIT alleles are damaged with resultant reduction or loss of the proapoptatic Fhit protein (refs. 6 and 7 for review), loss that has been correlated with worse outcome in some tumor types (8-11).The FHIT gene, in both mouse and human (12-15), encompasses a constitutive chromosomal fragile site, which is susceptible to DNA gaps and breaks on exposure to carcinogens, a susceptibility that explains the frequent alterations to the gene in preneoplastic and neoplastic lesions. Because the FHIT locus is so susceptible to damage and is inactivated early in many cancers and its loss may have prognostic significance in a large fraction of cancers, Fhit interacting proteins, substrates, and effectors are attractive targets for inclusion in cancer prevention and therapy strategies (16,17). To establish models for exploring the role of Fhit in cancer induction, prevention, and therapy, we have developed mouse strains carrying one or two inactivated Fhit alleles (18) and have studied the development of spontaneous and N-nitrosomethylbenzylamine (NMBA)-induced tumors in adult mice with intact or inactivated Fhit alleles. In a previous study, we had observed that 100% of Fhit ϩ͞Ϫ m...

The tumor suppressor gene FHIT spans a common fragile site and is highly susceptible to environmental carcinogens. FHIT inactivation and loss of expression is found in a large fraction of premaligant and malignant lesions. In this study, we were able to inhibit tumor development by oral gene transfer, using adenoviral or adenoassociated viral vectors expressing the human FHIT gene, in heterozygous Fhit ؉/؊ knockout mice, that are prone to tumor development after carcinogen exposure. We therefore suggest that FHIT gene therapy could be a novel clinical approach not only in treatment of early stages of cancer, but also in prevention of human cancer.

In recent years, studies of cancer development and recurrence have been influenced by the cancer stem cells (CSCs)/cancer-initiating cells (CICs) hypothesis. According to this, cancer is sustained by highly positioned, chemoresistant cells with extensive capacity of self renewal, which are responsible for disease relapse after chemotherapy. Growth of cancer cells as three-dimensional non-adherent spheroids is regarded as a useful methodology to enrich for cells endowed with CSC-like features. We have recently reported that cell cultures derived from malignant pleural effusions (MPEs) of patients affected by adenocarcinoma of the lung are able to efficiently form spheroids in non-adherent conditions supplemented with growth factors. By expression profiling, we were able to identify a set of genes whose expression is significantly upregulated in lung tumor spheroids versus adherent cultures. One of the most strongly upregulated gene was stearoyl-CoA desaturase (SCD1), the main enzyme responsible for the conversion of saturated into monounsaturated fatty acids. In the present study, we show both by RNA interference and through the use of a small molecule inhibitor that SCD1 is required for lung cancer spheroids propagation both in stable cell lines and in MPE-derived primary tumor cultures. Morphological examination and image analysis of the tumor spheroids formed in the presence of SCD1 inhibitors showed a different pattern of growth characterized by irregular cell aggregates. Electron microscopy revealed that the treated spheroids displayed several features of cellular damage and immunofluorescence analysis on optical serial sections showed apoptotic cells positive for the M30 marker, most of them positive also for the stemness marker ALDH1A1, thus suggesting that the SCD1 inhibitor is selectively killing cells with stem-like properties. Furthermore, SCD1-inhibited lung cancer cells were strongly impaired in their in vivo tumorigenicity and ALDH1A1 expression. These results suggest that SCD1 is a critical target in lung cancer tumor-initiating cells.

Recent evidences suggest that stearoyl-CoA-desaturase 1 (SCD1), the enzyme involved in monounsaturated fatty acids synthesis, has a role in several cancers. We previously demonstrated that SCD1 is important in lung cancer stem cells survival and propagation. In this article, we first show, using primary cell cultures from human lung adenocarcinoma, that the effectors of the Hippo pathway, Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), are required for the generation of lung cancer three-dimensional cultures and that SCD1 knock down and pharmacological inhibition both decrease expression, nuclear localization and transcriptional activity of YAP and TAZ. Regulation of YAP/TAZ by SCD1 is at least in part dependent upon β-catenin pathway activity, as YAP/TAZ downregulation induced by SCD1 blockade can be rescued by the addition of exogenous wnt3a ligand. In addition, SCD1 activation of nuclear YAP/TAZ requires inactivation of the β-catenin destruction complex. In line with the in vitro findings, immunohistochemistry analysis of lung adenocarcinoma samples showed that expression levels of SCD1 co-vary with those of β-catenin and YAP/TAZ. Mining available gene expression data sets allowed to observe that high co-expression levels of SCD1, β-catenin, YAP/TAZ and downstream targets have a strong negative prognostic value in lung adenocarcinoma. Finally, bioinformatics analyses directed to identify which gene combinations had synergistic effects on clinical outcome in lung cancer showed that poor survival is associated with high co-expression of SCD1, β-catenin and the YAP/TAZ downstream target birc5. In summary, our data demonstrate for the first time the involvement of SCD1 in the regulation of the Hippo pathway in lung cancer, and point to fatty acids metabolism as a key regulator of lung cancer stem cells.

Therapy of melanoma patients harboring activating mutations in the BRAF (V-raf murine sarcoma viral oncogene homolog B1) oncogene with a combination of BRAF and MEK inhibitors is plagued by the development of drug resistance. Mutational events, as well as adaptive mechanisms, contribute to the development of drug resistance. In this context we uncover here the role of a miRNA, miR-579-3p. We first show that low expression of miR-579-3p is a negative prognostic factor correlating with poor survival. Expression levels of miR-579-3p decrease from nevi to stage III/IV melanoma samples and even further in cell lines resistant to BRAF/MEK inhibitors. Mechanistically, we demonstrate that miR-579-3p acts as an oncosuppressor by targeting the 3′UTR of two oncoproteins: BRAF and an E3 ubiquitin protein ligase, MDM2. Moreover miR-579-3p ectopic expression impairs the establishment of drug resistance in human melanoma cells. Finally, miR-579-3p is strongly down-regulated in matched tumor samples from patients before and after the development of resistance to targeted therapies.miRNA | melanoma | targeted therapy | drug resistance

Cancer stem cells (CSCs) are an uncommon subset of tumor cells capable of self-renewal, differentiating, and recreating the parental tumor when transplanted into the murine background. Over the past two decades, efforts toward understanding CSC biology culminated into identifying a set of signaling pathways sustaining "stemness". Nevertheless, while metabolic rewiring is nowadays considered a hallmark of cancer, no consensus has been reached on the metabolic features underlying the plastic nature of CSCs, which are capable of residing in a dormant state, and able to rapidly proliferate when the need to repopulate the tumor mass arises. An emerging concept in the field of CSC metabolism is that these cells are extremely reliant on the activity of enzymes involved in lipid metabolism, such as stearoyl-CoA desaturase 1 (SCD1) and 3-hydroxy-3-methylglutharyl-coenzyme A reductase (HMG-CoAR). Indeed, SCD1 and HMG-CoAR have been described as key factors for the correct function of a number of concatenated pathways involved in CSC fate decision, such as Hippo and Wnt. In the present review, we describe metabolic futures of CSCs with a special focus on lipid metabolism, which until now represents an underappreciated force in maintaining CSCs and an attractive therapeutic target.

Malignant pleural effusions (MPEs) could represent an excellent source to culture a wide variety of cancer cells from different donors. In this study, we set up culture conditions for cancer cells deriving from MPEs of several patients affected by the most frequent form of lung cancer, namely the subset of non small cell lung cancers (NSCLC) classified as Lung Adenocarcinomas (AdenoCa) which account for approximately 40% of lung cancer cases. AdenoCa malignant pleural effusions gave rise to in vitro cultures both in adherent and/or in spheroid conditions in almost all cases analyzed. We characterized in greater detail two samples which showed the most efficient propagation in vitro. In these samples we also compared gene profiles of spheroid vs adherent cultures and identified a set of differentially expressed genes. Finally we achieved efficient tumor engraftment in recipient NOD/SCID mice, also upon inoculation of small number of cells, thus suggesting indirectly the presence of tumor initiating cells.