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1.  Cigarette smoke metabolically promotes cancer, via autophagy and premature aging in the host stromal microenvironment 
Cell Cycle  2013;12(5):818-825.
Cigarette smoke has been directly implicated in the disease pathogenesis of a plethora of different human cancer subtypes, including breast cancers. The prevailing view is that cigarette smoke acts as a mutagen and DNA damaging agent in normal epithelial cells, driving tumor initiation. However, its potential negative metabolic effects on the normal stromal microenvironment have been largely ignored. Here, we propose a new mechanism by which carcinogen-rich cigarette smoke may promote cancer growth, by metabolically “fertilizing” the host microenvironment. More specifically, we show that cigarette smoke exposure is indeed sufficient to drive the onset of the cancer-associated fibroblast phenotype via the induction of DNA damage, autophagy and mitophagy in the tumor stroma. In turn, cigarette smoke exposure induces premature aging and mitochondrial dysfunction in stromal fibroblasts, leading to the secretion of high-energy mitochondrial fuels, such as L-lactate and ketone bodies. Hence, cigarette smoke induces catabolism in the local microenvironment, directly fueling oxidative mitochondrial metabolism (OXPHOS) in neighboring epithelial cancer cells, actively promoting anabolic tumor growth. Remarkably, these autophagic-senescent fibroblasts increased breast cancer tumor growth in vivo by up to 4-fold. Importantly, we show that cigarette smoke-induced metabolic reprogramming of the fibroblastic stroma occurs independently of tumor neo-angiogenesis. We discuss the possible implications of our current findings for the prevention of aging-associated human diseases and, especially, common epithelial cancers, as we show that cigarette smoke can systemically accelerate aging in the host microenvironment. Finally, our current findings are consistent with the idea that cigarette smoke induces the “reverse Warburg effect,” thereby fueling “two-compartment tumor metabolism” and oxidative mitochondrial metabolism in epithelial cancer cells.
doi:10.4161/cc.23722
PMCID: PMC3610729  PMID: 23388463
carcinogens; cigarette smoke; cancer prevention; autophagy; senescence; premature aging; mitochondrial dysfunction; lactate; ketone bodies; breast cancer; tumor growth; microenvironment
2.  Creating a tumor-resistant microenvironment 
Cell Cycle  2013;12(3):480-490.
Here, we provide the necessary proof of concept, that it is possible to metabolically create a non-permissive or “hostile” stromal microenvironment, which actively prevents tumor engraftment in vivo. We developed a novel genetically engineered fibroblast cell line that completely prevents tumor formation in mice, with a 100% protection rate. No host side effects were apparent. This could represent a viable cellular strategy for preventing and treating a variety of human cancers. More specifically, we examined the autocrine and paracrine effects of the cellular delivery of TNFα on breast cancer tumor growth and cancer metabolism. For this purpose, we recombinantly overexpressed TNFα in human breast cancer cells (MDA-MB-231) or human immortalized fibroblasts (hTERT-BJ1). Our results directly show that TNFα functions as a potent tumor suppressor. Remarkably, TNFα-expressing breast cancer cells were viable, without any significant increases in their basal apoptotic rate. However, after 4 weeks post-implantation, TNFα-expressing breast cancer cells failed to form any tumors in xenografted mice (0 tumors/10 injections), ultimately conferring 100% protection against tumorigenesis. Similarly, TNFα-overexpressing fibroblasts were also viable, without any increases in apoptosis. Significantly, complete tumor suppression was obtained by co-injecting TNFα expressing stromal fibroblasts with human breast cancer cells, indicating that paracrine cell-mediated delivery of TNFα can also prevent tumor engraftment and growth (0 tumors/10 injections). Mechanistically, TNFα induced autophagy and mitochondrial dysfunction in both epithelial cancer cells and stromal fibroblasts, preventing energy transfer from the tumor microenvironment, likely “starving” the cancer cells to death. In addition, via qRT-PCR analysis of MDA-MB-231 cells, we observed that TNFα mediated the upregulation of gene transcripts associated with inflammation and senescence [IL-1-β, IL-6, IL-8, MCP-1, COX-2, p21(WAF1/CIP1)] and downregulated known tumor-promoting genes (collagen VI and MMP2). Recombinant overexpression of TNFα receptor(s) in MDA-MB-231 cells also significantly reduced tumor growth, but was not as effective as the TNFα ligand itself in preventing tumor growth. Thus, we propose that stromal cell-mediated delivery of TNFα to human tumors [using transfected fibroblasts or mesenchymal stem cells (hMSCs)] may be a novel and effective strategy for the prevention and treatment of human cancers.
doi:10.4161/cc.23370
PMCID: PMC3587449  PMID: 23292149
tumor necrosis factor (TNF); cancer prevention; cellular therapy; fibroblast mediated delivery; mitochondrial dysfunction; breast cancer; tumor growth; tumor cell engraftment; autophagy; apoptosis
3.  The KIT Exon 11 Stop Codon Mutation in Gastrointestinal Stromal Tumors: What Is the Clinical Meaning? 
Gut and Liver  2012;7(1):35-40.
Background/Aims
Gastrointestinal stromal tumors (GISTs) strongly express a receptor tyrosine kinase (RTK, c-KIT-CD117) harboring a KIT mutation that causes constitutive receptor activation leading to the development and growth of tumors; 35% of GISTs without KIT mutations have platelet-derived growth factor receptor alpha (PDGFRA) mutations, and the type of mutation plays an important role in the response to treatment. This study aimed to establish the frequency of stop codon mutations in the RTKs, KIT, and PDGFRA, in GISTs and correlate this molecular alteration with protein expression and treatment responsiveness.
Methods
Seventy-nine GISTs were analyzed for both KIT and PDGFRA mutations. Immunohistochemical expression was studied in tissue microarray blocks.
Results
We found three rare KIT mutations in exon 11 that induced a stop codon, two at position 563 and one at position 589, which have never been described before. All three tumors were CD117-, DOG1-, and CD34-positive. Two patients with a KIT stop codon mutation did not respond to imatinib therapy and died shortly after treatment.
Conclusions
The association between stop codon mutations in KIT and patient survival, if confirmed in a larger population, may be useful in choosing effective therapies.
doi:10.5009/gnl.2013.7.1.35
PMCID: PMC3572318  PMID: 23423603
Gastrointestinal stromal tumors; c-KIT; Platelet-derived growth factor alpha receptor
4.  Association between HRAS rs12628 and rs112587690 polymorphisms with the risk of melanoma in the North American population 
HRAS belongs to the RAS genes superfamily. RAS genes are important players in several human tumors and the single-nucleotide polymorphism rs12628 has been shown to contribute to the risk of bladder, colon, gastrointestinal, oral, and thyroid carcinoma. We hypothesized that this SNP may affect the risk of cutaneous melanoma as well. HRAS gene contains a polymorphic region (rs112587690), a repeated hexanucleotide -GGGCCT-located in intron 1. Three alleles of this region, P1, P2, and P3, have been identified that contain two, three, and four repeats of the hexanucleotide, respectively. We investigated the clinical impact of these polymorphisms in a case–control study. A total of 141 melanoma patients and 118 healthy donors from the North America Caucasian population were screened for rs12628 and rs112587690 polymorphisms. Genotypes were assessed by capillary sequencing or fragment analysis, respectively, and rs12628 CC and rs112587690 P1P1 genotypes significantly associated with increased melanoma risk (OR = 3.83, p = 0.003; OR = 11.3, p = 0.033, respectively), while rs112587690 P1P3 frequency resulted significantly higher in the control group (OR = 0.5, p = 0.017). These results suggest that rs12628 C homozygosis may be considered a potential risk factor for melanoma development in the North American population possibly through the linkage to rs112587690.
doi:10.1007/s12032-012-0255-3
PMCID: PMC3505523  PMID: 22618666
HRAS; Polymorphism; Melanoma; rs12628; rs112587690
5.  Caveolin-1 promotes pancreatic cancer cell differentiation and restores membranous E-cadherin via suppression of the epithelial-mesenchymal transition 
Cell Cycle  2011;10(21):3692-3700.
Pancreatic cancer is one of the deadliest cancers due to early rapid metastasis and chemoresistance. Recently, epithelial to mesenchymal transition (EMT) was shown to play a key role in the pathogenesis of pancreatic cancer. To understand the role of caveolin-1 (Cav-1) in EMT, we overexpressed Cav-1 in a pancreatic cancer cell line, Panc 10.05, that does not normally express Cav-1. Here, we show that Cav-1 expression in pancreatic cancer cells induces an epithelial phenotype and promotes cell-cell contact, with increased expression of plasma membrane bound E-cadherin and β-catenin. Mechanistically, Cav-1 induces Snail downregulation and decreased activation of AKT, MAPK and TGFβ-Smad signaling pathways. In vitro, Cav-1 expression reduces cell migration and invasion, and attenuates doxorubicin-chemoresistance of pancreatic cancer cells. Importantly, in vivo studies revealed that Cav-1 expression greatly suppresses tumor formation in a xenograft model. Most interestingly, Panc/Cav-1 tumors displayed organized nests of differentiated cells that were totally absent in control tumors. Confirming our in vitro results, these nests of differentiated cells showed reexpression of E-cadherin and β-catenin at the cell membrane. Thus, we provide evidence that Cav-1 functions as a crucial modulator of EMT and cell differentiation in pancreatic cancer.
doi:10.4161/cc.10.21.17895
PMCID: PMC3266007  PMID: 22041584
caveolae; caveolin-1; epithelial-mesenchymal transition; E-cadherin; pancreatic cancer; cell differentiation; chemoresistance
6.  A molecular computational model improves the preoperative diagnosis of thyroid nodules 
BMC Cancer  2012;12:396.
Background
Thyroid nodules with indeterminate cytological features on fine needle aspiration (FNA) cytology have a 20% risk of thyroid cancer. The aim of the current study was to determine the diagnostic utility of an 8-gene assay to distinguish benign from malignant thyroid neoplasm.
Methods
The mRNA expression level of 9 genes (KIT, SYNGR2, C21orf4, Hs.296031, DDI2, CDH1, LSM7, TC1, NATH) was analysed by quantitative PCR (q-PCR) in 93 FNA cytological samples. To evaluate the diagnostic utility of all the genes analysed, we assessed the area under the curve (AUC) for each gene individually and in combination. BRAF exon 15 status was determined by pyrosequencing. An 8-gene computational model (Neural Network Bayesian Classifier) was built and a multiple-variable analysis was then performed to assess the correlation between the markers.
Results
The AUC for each significant marker ranged between 0.625 and 0.900, thus all the significant markers, alone and in combination, can be used to distinguish between malignant and benign FNA samples. The classifier made up of KIT, CDH1, LSM7, C21orf4, DDI2, TC1, Hs.296031 and BRAF had a predictive power of 88.8%. It proved to be useful for risk stratification of the most critical cytological group of the indeterminate lesions for which there is the greatest need of accurate diagnostic markers.
Conclusion
The genetic classification obtained with this model is highly accurate at differentiating malignant from benign thyroid lesions and might be a useful adjunct in the preoperative management of patients with thyroid nodules.
doi:10.1186/1471-2407-12-396
PMCID: PMC3503705  PMID: 22958914
Thyroid; Fine-needle aspiration (FNA); Area under the curve (AUC); Computational model; Preoperative diagnosis
7.  Association between HRAS rs12628 and rs112587690 polymorphisms with the risk of melanoma in the North American population 
HRAS belongs to the RAS genes superfamily. RAS genes are important players in several human tumors and the single-nucleotide polymorphism rs12628 has been shown to contribute to the risk of bladder, colon, gastrointestinal, oral, and thyroid carcinoma. We hypothesized that this SNP may affect the risk of cutaneous melanoma as well. HRAS gene contains a polymorphic region (rs112587690), a repeated hexanucleotide -GGGCCT- located in intron 1. Three alleles of this region, P1, P2, and P3, have been identified that contain two, three, and four repeats of the hexanucleotide, respectively. We investigated the clinical impact of these polymorphisms in a case–control study. A total of 141 melanoma patients and 118 healthy donors from the North America Caucasian population were screened for rs12628 and rs112587690 polymorphisms. Genotypes were assessed by capillary sequencing or fragment analysis, respectively, and rs12628 CC and rs112587690 P1P1 genotypes significantly associated with increased melanoma risk (OR = 3.83, p = 0.003; OR = 11.3, p = 0.033, respectively), while rs112587690 P1P3 frequency resulted significantly higher in the control group (OR = 0.5, p = 0.017). These results suggest that rs12628 C homozygosis may be considered a potential risk factor for melanoma development in the North American population possibly through the linkage to rs112587690.
doi:10.1007/s12032-012-0255-3
PMCID: PMC3505523  PMID: 22618666
HRAS; Polymorphism; Melanoma; rs12628; rs112587690
8.  c-KIT receptor expression is strictly associated with the biological behaviour of thyroid nodules 
Background
A large amount of information has been collected on the molecular tumorigenesis of thyroid cancer. A low expression of c-KIT gene has been reported during the transformation of normal thyroid epithelium to papillary carcinoma suggesting a possible role of the gene in the differentiation of thyroid tissue rather than in the proliferation. The initial presentation of thyroid carcinoma is through a nodule and the best way nowadays to evaluate it is by fine-needle aspiration (FNA). However many thyroid FNAs are not definitively benign or malignant, yielding an indeterminate or suspicious diagnosis which ranges from 10 to 25% of FNAs. BRAF mutational analysis is commonly used to assess the malignancy of thyroid nodules but unfortunately it still leaves indeterminate diagnoses. The development of molecular initial diagnostic tests for evaluating a thyroid nodule is needed in order to define optimal surgical approach for patients with uncertain diagnosis pre- and intra-operatively.
Methods
In this study we extracted RNA from 82 FNA smears, 46 malignant and 36 benign at the histology, in order to evaluate by quantitative Real Time PCR the expression levels of c-KIT gene.
Results
We have found a highly preferential decrease rather than increase in transcript of c-KIT in malignant thyroid lesions compared to the benign ones. To explore the diagnostic utility of c-KIT expression in thyroid nodules, its expression values were divided in four arbitrarily defined classes, with class I characterized by the complete silencing of the gene. Class I and IV represented the two most informative groups, with 100% of the samples found malignant or benign respectively. The molecular analysis was proven by ROC (receiver operating characteristic) analysis to be highly specific and sensitive improving the cytological diagnostic accuracy of 15%.
Conclusion
We propose the use of BRAF test (after uncertain cytological diagnosis) to assess the malignancy of thyroid nodules at first, then the use of the c-KIT expression to ultimately assess the diagnosis of the nodules that otherwise would remain suspicious. The c-KIT expression-based classification is highly accurate and may provide a tool to overcome the difficulties in today's preoperative diagnosis of thyroid suspicious malignancies.
doi:10.1186/1479-5876-10-7
PMCID: PMC3267663  PMID: 22233760
c-KIT; thyroid cancer; FNAC
9.  Down-Regulation of Serum/Glucocorticoid Regulated Kinase 1 in Colorectal Tumours Is Largely Independent of Promoter Hypermethylation 
PLoS ONE  2010;5(11):e13840.
Background
We have previously shown that serum/glucocorticoid regulated kinase 1 (SGK1) is down-regulated in colorectal cancers (CRC) with respect to normal tissue. As hyper-methylation of promoter regions is a well-known mechanism of gene silencing in cancer, we tested whether the SGK1 promoter region was methylated in colonic tumour samples.
Methodology/Principal Findings
We investigated the methylation profile of the two CpG islands present in the promoter region of SGK1 in a panel of 5 colorectal cancer cell lines by sequencing clones of bisulphite-treated DNA samples. We further confirmed our findings in a panel of 10 normal and 10 tumour colonic tissue samples of human origin. We observed CpG methylation only in the smaller and more distal CpG island in the promoter region of SGK1 in both normal and tumour samples of colonic origin. We further identified a single nucleotide polymorphism (SNP, rs1743963) which affects methylation of the corresponding CpG.
Conclusions/Significance
Our results show that even though partial methylation of the promoter region of SGK1 is present, this does not account for the different expression levels seen between normal and tumour tissue.
doi:10.1371/journal.pone.0013840
PMCID: PMC2974649  PMID: 21079778
11.  The role of the pathologist in tissue banking: European Consensus Expert Group Report 
Virchows Archiv  2010;456(4):449-454.
Human tissue biobanking encompasses a wide range of activities and study designs and is critical for application of a wide range of new technologies (-“omics”) to the discovery of molecular patterns of disease and for implementation of novel biomarkers into clinical trials. Pathology is the cornerstone of hospital-based tissue biobanking. Pathologists not only provide essential information identifying the specimen but also make decisions on what should be biobanked, making sure that the timing of all operations is consistent with both the requirements of clinical diagnosis and the optimal preservation of biological products. This document summarizes the conclusions of a Pathology Expert Group Meeting within the European Biological and Biomolecular Research Infrastructure (BBMRI) Program. These recommendations are aimed at providing guidance for pathologists as well as for institutions hosting biobanks on how to better integrate and support pathological activities within the framework of biobanks that fulfill international standards.
doi:10.1007/s00428-010-0887-7
PMCID: PMC2852521  PMID: 20157825
Pathology; Biobanks; Biomarkers; Harmonization; Standards; Translational research
12.  A novel breast cancer-associated BRIP1 (FANCJ/BACH1) germ line mutation impairs protein stability and function 
Purpose
BRIP1 (FANCJ/BACH1), which encodes a DNA helicase that interacts with BRCA1, has been suggested to be a low penetrance breast cancer predisposing gene. We aimed to assess whether BRIP1 mutations contribute to breast cancer susceptibility in our population and, if so, to investigate the impact of such mutation(s) on BRIP1 function.
Experimental Design
A series of 49 breast/ovarian cancer families, devoid of a BRCA1/BRCA2 mutation, were screened for BRIP1 mutations. Functional analyses, including co-immunoprecipitation and stability assays, were employed to further characterize a previously unreported variant.
Results
Five sequence alterations were identified, of which four had been already described. Herein, we report a novel BRIP1 germ line mutation identified in a woman with early onset breast cancer. The mutation consists of a four-nucleotide deletion (c.2992-2995delAAGA) in BRIP1 exon 20 that causes a shift in the reading frame, disrupts the BRCA1-binding domain of BRIP1 and creates a premature stop codon. Functional analysis of the recombinant mutant protein in transfected cells showed that the truncation interferes with the stability of the protein and with its ability to interact with BRCA1. Loss of the wild type BRIP1 allele with retention of the mutated one was observed in the patient’s breast tumor tissue.
Conclusions
These results, by showing that the newly identified BRIP1 c.2992-2995delAAGA mutation is associated with instability and functional impairment of the encoded protein, provide further evidence of a breast cancer-related role for BRIP1.
doi:10.1158/1078-0432.CCR-08-0087
PMCID: PMC2561321  PMID: 18628483
BRIP1; breast cancer susceptibility; BRCA1; tumor suppressor genes; Fanconi anaemia
13.  Lymphoepithelioma-like hepatocellular carcinoma: A case report and a review of the literature 
Lymphoepithelioma is a particular form of undifferentiated carcinoma, characterized by a prominent lymphoid stroma, originally described in the nasopharynx. Lymphoid stroma-rich carcinomas arising in other organs have been termed lymphoepithelioma-like carcinoma (LELC). In the liver, primary LELCs are very rare, and the majority has been identified as cholangiocarcinomas. Here a rare case of lymphoepithelioma-like hepatocellular carcinoma (HCC) is described. A 47-year old woman presented with abdominal pain. Ultrasonography revealed a liver nodule, 2.2 cm in diameter, localized in the right lobe, adjacent to the gallbladder. Viral markers for hepatic B virus (HBV), hepatic C virus (HCV) and Epstein-Barr virus (EBV) were negative. The nodule was hypoechogenic. The patient underwent surgery, with resection of the nodule. Histology showed hepatocellular carcinoma, characterized by a prominent lymphoid infiltrate. At immunocytochemistry, tumor cells were reactive for Hep Par1 and glypican 3. Immunophenotyping of tumor infiltrating lymphocytes evidenced the predominance of CD8+ cytotoxic suppressor T cells. The postoperative clinical outcome was favorable and the patient was recurrence-free 15 mo after resection. This case, to the best of our knowledge, is the first reported non EBV and non cirrhosis-associated lymphoepithelioma-like hepatocellular carcinoma. The association between the lack of EBV infection, the absence of cirrhosis, a “cytotoxic profile” of the inflammatory infiltrate and a good prognosis could identify a variant of lymphoepithelioma-like HCC with a favorable clinical outcome.
doi:10.3748/wjg.14.4694
PMCID: PMC2738796  PMID: 18698686
Liver; Hepatocellular carcinoma; Tumor infiltrating lymphocytes; Primary liver tumors; Liver lymphoepithelioma

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