The Viral Origin of Human Breast Cancer: From the Mouse Mammary Tumor Virus (MMTV) to the Human Betaretrovirus (HBRV).

A Human Betaretrovirus (HBRV) has been identified in humans, dating as far back as about 4500 years ago, with a high probability of it being acquired by our species around 10,000 years ago, following a species jump from mice to humans. HBRV is the human homolog of the MMTV (mouse mammary tumor virus), which is the etiological agent of murine mammary tumors. The hypothesis of a HMTV (human mammary tumor virus) was proposed about 50 years ago, and has acquired a solid scientific basis during the last 30 years, with the demonstration of a robust link with breast cancer and with PBC, primary biliary cholangitis. This article summarizes most of what is known about MMTV/HMTV/HBRV since the discovery of MMTV at the beginning of last century, to make evident both the quantity and the quality of the research supporting the existence of HBRV and its pathogenic role. Here, it is sufficient to mention that scientific evidence includes that viral sequences have been identified in breast-cancer samples in a worldwide distribution, that the complete proviral genome has been cloned from breast cancer and patients with PBC, and that saliva contains HBRV, as a possible route of inter-human infection. Controversies that have arisen concerning results obtained from human tissues, many of them outdated by new scientific evidence, are critically discussed and confuted.

A human MMTV-like betaretrovirus linked to breast cancer has been present in humans at least since the copper age.

The betaretrovirus Mouse Mammary Tumor Virus (MMTV) is the well characterized etiological agent of mammary tumors in mice. In contrast, the etiology of sporadic human breast cancer (BC) is unknown, but accumulating data indicate a possible viral origin also for these malignancies. The presence of MMTVenv-like sequences (MMTVels) in the human salivary glands and saliva supports the latter as possible route of inter-human dissemination. In the absence of the demonstration of a mouse-man transmission of MMTV, we considered the possibility that a cross-species transmission could have occurred in ancient times. Therefore, we investigated MMTVels in the ancient dental calculus, which originates from saliva and is an excellent material for paleovirology. The calculus was collected from 36 ancient human skulls, excluding any possible mouse contamination. MMTV-like sequences were identified in the calculus of 6 individuals dated from the Copper Age to the 17th century. The MMTV-like sequences were compared with known human endogenous betaretroviruses and with animal exogenous betaretroviruses, confirming their exogenous origin and relation to MMTV. These data reveal that a human exogenous betaretrovirus similar to MMTV has existed at least since 4,500 years ago and indirectly support the hypothesis that it could play a role in human breast cancer.

Mouse mammary tumor virus (MMTV) - like exogenous sequences are associated with sporadic but not hereditary human breast carcinoma.

The inheritance of mutated suppressor genes, such as BRCA1 and BRCA2, is acknowledged as an etiological factor in hereditary breast carcinoma (HBC). Two different molecular mechanisms are possible; the Knudson's "two hits" or the gene haploinsufficiency. Etiology of sporadic breast carcinoma (SBC) is not known, although data support the possible role of the betaretrovirus Mouse Mammary Tumor Virus (MMTV). This study analyzes the presence of MMTV exogenous sequences in two representative groups of HBC and SBC, excluding any contamination by murine and retroviral material and endogenous betaretroviruses. The 30.3% of 56 SBC contained MMTV sequences, against the 4.2% of 47 HBC (p < 0.001). Cases positive for viral sequences showed the presence of p14, signal peptide of the MMTV envelope precursor. This result was expected based on the fact that HBCs, having a specific genetic etiology, do not need the action of a carcinogenetic viral agent. Moreover, the striking results obtained by comparing two groups of vastly different tumors represent an additional element of quality control: the distinction between HBC and SBC is so well-defined that results cannot be ascribed to mere coincidence. This paper strengthens the hypothesis for a viral etiology for human sporadic breast carcinoma.

Cancer astrocytes have a more conserved molecular status in long recurrence free survival (RFS) IDH1 wild-type glioblastoma patients: new emerging cancer players.

Glioblastoma is a devastating disease that despite all the information gathered so far, its optimal management remains elusive due to the absence of validated targets from clinical studies. A better clarification of the molecular mechanisms is needed. In this study, having access to IDH1 wild-type glioblastoma of patients with exceptionally long recurrence free survival (RFS), we decided to compare their mutational and gene expression profile to groups of IDH1 wild-type glioblastoma of patients with shorter RFS, by using NGS technology. The exome analysis revealed that Long-RFS tumors have a lower mutational rate compared to the other groups. A total of 158 genes were found differentially expressed among the groups, 112 of which distinguished the two RFS extreme groups. Overall, the exome data suggests that shorter RFS tumors could be, chronologically, in a more advanced state in the muli-step tumor process of sequential accumulation of mutations. New players in this kind of cancer emerge from the analysis, confirmed at the RNA/DNA level, identifying, therefore, possible oncodrivers or tumor suppressor genes.

Polymorphisms and mutations in GSTP1, RAD51, XRCC1 and XRCC3 genes in breast cancer patients.

BACKGROUND: Genotoxic factors, including ionizing radiation and oxidative stress, are associated with genomic instability and development of breast cancer (BC). The homologous recombination DNA repair (HRR) pathway, base excision repair (BER) mechanism, and antioxidative enzymes are required as defense mechanisms against these DNA damaging agents. GSTP1, XRCC1, XRCC3 and RAD51 proteins are essential components of antioxidation, BER and HRR of DNA, respectively. Deficiencies in BER, HRR and antioxidation pathways are involved in the progression of cancer. METHODS: Genomic DNA was extracted from formalin-fixed, paraffin-embedded tissue and blood samples of BC patients of an Italian population. Genomic DNA was also extracted from blood specimens of a control group. DNA sequencing was performed for six single-nucleotide polymorphisms (SNPs) in the GSTP1, RAD51, XRCC1 and XRCC3 genes in BC patients and the control group. RESULTS: Two variants in the 5'-UTR of the XRCC3 (rs1799794 A/G) and RAD51 (rs1801321) genes showed a significant association with susceptibility to BC (OR = 4.125; 95% CI 1.057-16.102; p = 0.03 and OR = 2.04; 95% CI 0.4925-8.449; p = 0.007, respectively). Additionally, we reported 2 mutations in intron 7 of the XRCC3 gene, CTdel (rs543072564) and A/G (rs369703243). CONCLUSIONS: Our results underscored the existence of an association between XRCC3-5'-UTR-A/G (rs1799794) and RAD51-5'-UTR G172T (rs1801321) genotypes and BC risk in an Italian population. The presence of mutations in the intronic region of the XRCC3 gene highlights the importance of more sequence screening of DNA repair genes for possible genetic penetrance in BC.

Erratum to: Mouse mammary tumor-like virus (MMTV) is present in human breast tissue before development of virally associated breast cancer.

[This corrects the article DOI: 10.1186/s13027-016-0113-6.].

Mouse mammary tumor-like virus (MMTV) is present in human breast tissue before development of virally associated breast cancer.

BACKGROUND: There is substantial evidence that a virus homologous to mouse mammary tumor virus (MMTV) may have a role in human breast cancer. The present study indicates that those who developed breast cancer associated with an MMTV-like virus had this virus in their non-cancerous breast tissues years before the cancer developed. METHODS: Polymerase chain reaction (PCR) techniques and sequencing were used to identify MMTV-like envelope gene sequences (MMTV-like env sequences) in Australian benign breast biopsy specimens from women who several years later developed breast cancer. Murine contamination was excluded by stringent laboratory procedures, and the absence of intracisternal A particle sequences and mitochondrial cyclooxygenase sequences. RESULTS: MMTV-like env sequences (also called HMTV sequences to denote their source) were found in 9 of 25 breast cancer specimens (36%). Among 25 non-cancerous breast biopsies of these same patients taken 1 to 11 years earlier, six contained MMTV-like sequences (24%). Five of the six were among the nine virally-associated breast cancers. In two pairs of specimens, benign and malignant, env sequences were 97% identical. CONCLUSIONS: The identification of MMTV (MMTV-like) sequences in breast tissues prior to the development of MMTV positive breast cancer fulfills a key criterion for a possible causal role for the MMTV-like virus in human breast cancer.

A new immunization and treatment strategy for mouse mammary tumor virus (MMTV) associated cancers.

Mouse Mammary Tumor Virus (MMTV) causes mammary carcinoma or lymphoma in mice. An increasing body of evidence in recent years supports its involvement also in human sporadic breast cancer. It is thus of importance to develop new strategies to impair the development, growth and metastasis of MMTV-associated cancers. The signal peptide of the envelope precursor protein of this virus: MMTV-p14 (p14) is an excellent target for such strategies, due to unique characteristics distinct from its regular endoplasmic reticulum targeting function. These include cell surface expression in: murine cancer cells that harbor the virus, human breast cancer (MCF-7) cells that ectopically express p14, as well as cultured human cells derived from an invasive ductal breast carcinoma positive for MMTV sequences. These findings support its use in signal peptide-based immune targeting. Indeed, priming and boosting mice with p14 elicits a specific anti-signal peptide immune response sufficient for protective vaccination against MMTV-associated tumors. Furthermore, passive immunization using a combination of anti-p14 monoclonal antibodies or the transfer of T-cells from immunized mice (Adoptive Cell Transfer) is also therapeutically effective. With reports demonstrating involvement of MMTV in human breast cancer, we propose the immune-mediated targeting of p14 as a strategy for prevention, treatment and diagnosis of MMTV-associated cancers.

Homozygous T172T and Heterozygous G135C Variants of Homologous Recombination Repairing Protein RAD51 are Related to Sporadic Breast Cancer Susceptibility.

Breast cancer (BC) is the most common cancer and the second leading cause of death among women worldwide. Only 10% of BC cases have been related to genetic predisposition. Rad51, a homologous recombination (HR) protein plays an important role in HR in meiosis and repairing DNA double-strand breaks. Expression of RAD51 may be a predictive biomarker in certain types of cancers. The exact mechanisms involved in the regulation of RAD51 expression are not fully understood, but certain transcription factors have been suggested to be the tuning mechanism of its expression. In this study, we propose that polymorphisms in the 5'-UTR promoter region of the RAD51 gene are prognostic factors for BC development. Direct sequencing of 106 samples from sporadic BC patients and 54 samples from a control group was performed. FFPE samples were the choice of sample collection, which might be a limitation of our study. Homologous variant T172T alone was found to be significantly associated with BC risk (OR 3.717, 95% CI 2.283-6.052, p < 0.0001). On the other hand, heterozygous G135C did not show any significant relationship with risk of sporadic BC (OR 1.598, 95% CI 0.5638-4.528, p > 0.05). Moreover, both variants; homozygous T172T and heterozygous G135C together; showed a significant relationship with sporadic BC susceptibility.

The combination of four molecular markers improves thyroid cancer cytologic diagnosis and patient management.

BACKGROUND: Papillary thyroid cancer is the most common endocrine malignancy. The most sensitive and specific diagnostic tool for thyroid nodule diagnosis is fine-needle aspiration (FNA) biopsy with cytological evaluation. Nevertheless, FNA biopsy is not always decisive leading to "indeterminate" or "suspicious" diagnoses in 10%-30% of cases. BRAF V600E detection is currently used as molecular test to improve the diagnosis of thyroid nodules, yet it lacks sensitivity. The aim of the present study was to identify novel molecular markers/computational models to improve the discrimination between benign and malignant thyroid lesions. METHODS: We collected 118 pre-operative thyroid FNA samples. All 118 FNA samples were characterized for the presence of the BRAF V600E mutation (exon15) by pyrosequencing and further assessed for mRNA expression of four genes (KIT, TC1, miR-222, miR-146b) by quantitative polymerase chain reaction. Computational models (Bayesian Neural Network Classifier, discriminant analysis) were built, and their ability to discriminate benign and malignant tumors were tested. Receiver operating characteristic (ROC) analysis was performed and principal component analysis was used for visualization purposes. RESULTS: In total, 36/70 malignant samples carried the V600E mutation, while all 48 benign samples were wild type for BRAF exon15. The Bayesian neural network (BNN) and discriminant analysis, including the mRNA expression of the four genes (KIT, TC1, miR-222, miR-146b) showed a very strong predictive value (94.12% and 92.16%, respectively) in discriminating malignant from benign patients. The discriminant analysis showed a correct classification of 100% of the samples in the malignant group, and 95% by BNN. KIT and miR-146b showed the highest diagnostic accuracy of the ROC curve, with area under the curve values of 0.973 for KIT and 0.931 for miR-146b. CONCLUSIONS: The four genes model proposed in this study proved to be highly discriminative of the malignant status compared with BRAF assessment alone. Its implementation in clinical practice can help in identifying malignant/benign nodules that would otherwise remain suspicious.

Mitochondrial mass, a new metabolic biomarker for stem-like cancer cells: Understanding WNT/FGF-driven anabolic signaling.

Here, we developed an isogenic cell model of "stemness" to facilitate protein biomarker discovery in breast cancer. For this purpose, we used knowledge gained previously from the study of the mouse mammary tumor virus (MMTV). MMTV initiates mammary tumorigenesis in mice by promoter insertion adjacent to two main integration sites, namely Int-1 (Wnt1) and Int-2 (Fgf3), which ultimately activates Wnt/ß-catenin signaling, driving the propagation of mammary cancer stem cells (CSCs). Thus, to develop a humanized model of MMTV signaling, we over-expressed WNT1 and FGF3 in MCF7 cells, an ER(+) human breast cancer cell line. We then validated that MCF7 cells over-expressing both WNT1 and FGF3 show a 3.5-fold increase in mammosphere formation, and that conditioned media from these cells is also sufficient to promote stem cell activity in untransfected parental MCF7 and T47D cells, as WNT1 and FGF3 are secreted factors. Proteomic analysis of this model system revealed the induction of i) EMT markers, ii) mitochondrial proteins, iii) glycolytic enzymes and iv) protein synthesis machinery, consistent with an anabolic CSC phenotype. MitoTracker staining validated the expected WNT1/FGF3-induced increase in mitochondrial mass and activity, which presumably reflects increased mitochondrial biogenesis. Importantly, many of the proteins that were up-regulated by WNT/FGF-signaling in MCF7 cells, were also transcriptionally over-expressed in human breast cancer cells in vivo, based on the bioinformatic analysis of public gene expression datasets of laser-captured patient samples. As such, this isogenic cell model should accelerate the discovery of new biomarkers to predict clinical outcome in breast cancer, facilitating the development of personalized medicine.Finally, we used mitochondrial mass as a surrogate marker for increased mitochondrial biogenesis in untransfected MCF7 cells. As predicted, metabolic fractionation of parental MCF7 cells, via MitoTracker staining, indicated that high mitochondrial mass is a new metabolic biomarker for the enrichment of anabolic CSCs, as functionally assessed by mammosphere-forming activity. This observation has broad implications for understanding the role of mitochondrial biogenesis in the propagation of stem-like cancer cells. Technically, this general metabolic approach could be applied to any cancer type, to identify and target the mitochondrial-rich CSC population.The implications of our work for understanding the role of mitochondrial metabolism in viral oncogenesis driven by random promoter insertions are also discussed, in the context of MMTV and ALV infections.

Human saliva as route of inter-human infection for mouse mammary tumor virus.

Etiology of human breast cancer is unknown, whereas the Mouse Mammary Tumor Virus (MMTV) is recognized as the etiologic agent of mouse mammary carcinoma. Moreover, this experimental model contributed substantially to our understanding of many biological aspects of the human disease. Several data strongly suggest a causative role of MMTV in humans, such as the presence of viral sequences in a high percentage of infiltrating breast carcinoma and in its preinvasive lesions, the production of viral particles in primary cultures of breast cancer, the ability of the virus to infect cells in culture. This paper demonstrates that MMTV is present in human saliva and salivary glands. MMTV presence was investigated by fluorescent PCR, RT-PCR, FISH, immunohistochemistry, and whole transcriptome analysis. Saliva was obtained from newborns, children, adults, and breast cancer patients. The saliva of newborns is MMTV-free, whereas MMTV is present in saliva of children (26.66%), healthy adults (10.60%), and breast cancer patients (57.14% as DNA and 33.9% as RNA). MMTV is also present in 8.10% of salivary glands. RNA-seq analysis performed on saliva of a breast cancer patient demonstrates a high expression of MMTV RNA in comparison to negative controls. The possibility of a contamination by murine DNA was excluded by murine mtDNA and IAP LTR PCR. These findings confirm the presence of MMTV in humans, strongly suggest saliva as route in inter-human infection, and support the hypothesis of a viral origin for human breast carcinoma.

Investigating molecular alterations to profile short- and long-term recurrence-free survival in patients with primary glioblastoma.

Glioblastoma (GB) is the most aggressive type of primary brain tumor. Despite the progress in recent years regarding the diagnosis and treatment of GB, the recurrence rate remains high, due to the infiltrative and dispersive nature of the tumor, which typically results in poor patient prognosis. In the present study, 19 formalin-fixed, paraffin-embedded GB samples were selected from patients with GB tumors. The samples were classified into a short or long recurrence-free survival (RFS) group, based on the time of first recurrence of the disease in the patients. The 19 samples were molecularly characterized for mutations in the isocitrate dehydrogenase 1 (IDH1) gene, amplification of the epidermal growth factor receptor (EGFR) gene, presence of the EGFR variant III, and methylation of the promoter region of the O6-methylguanine-DNA methyltransferase (MGMT) gene. Then, the expression of 84 genes involved in cell-cell and cell-matrix interactions, and that of 84 microRNAs (miRNAs) associated with brain cancer, was profiled. In addition, a copy number variation analysis of 23 genes reported to undergo frequent genomic alterations in human glioma was also performed. Differences in the expression levels of a number of genes were detected across the short and long RFS groups. Among these genes, 5 in particular were selected, and a 5-genes combination approach was developed, which was able to differentiate between patients with short and long RFS outcome. The high levels of sensitivity and precision displayed by this 5-genes combination approach, which were confirmed with a cross-validation method, provide a strong foundation for further validation of the involvement of the aforementioned genes in GB in a larger patient population. In conclusion, the present study has demonstrated how the expression pattern of miRNAs and mRNAs in patients with GB defines a particular molecular hallmark that may increase or reduce the aggressive behavior of GB tumors, thus influencing the survival rates of patients with GB, their response to therapy and their tendency to suffer a relapse.

Accessibilome of human glioblastoma: collagen-VI-alpha-1 is a new target and a marker of poor outcome.

Functional targeted therapy has unfortunately failed to improve the outcome of glioblastoma patients. Success stories evidenced by the use of antibody-drug conjugates in other tumor types are encouraging, but targets specific to glioblastoma and accessible through the bloodstream remain scarce. In the current work, we have identified and characterized novel and accessible proteins using an innovative proteomic approach on six human glioblastomas; the corresponding data have been deposited in the PRIDE database identifier PXD001398. Among several clusters of uniquely expressed proteins, we highlight collagen-VI-alpha-1 (COL6A1) as a highly expressed tumor biomarker with low levels in most normal tissues. Immunohistochemical analysis of glioma samples from 61 patients demonstrated that COL6A1 is a significant and consistent feature of high-grade glioma. Deposits of COL6A1 were evidenced in the perivascular regions of the tumor-associated vasculature and in glioma cells found in pseudopalisade structures. Retrospective analysis of public gene-expression data sets from over 300 glioma patients demonstrated a significant correlation of poor patient outcome and high COL6A1 expression. In a proof-of-concept study, we use chicken chorioallantoic membrane in vivo model to show that COL6A1 is a reachable target for IV-injected antibodies. The present data warrant further development of human COL6A1 antibodies for assessing the quantitative biodistribution in the preclinical tumor models.

VHL and HIF-1α: gene variations and prognosis in early-stage clear cell renal cell carcinoma.

Von Hipple-Lindau gene (VHL) inactivation represents the most frequent abnormality in clear cell renal cell carcinoma (ccRCC). Hypoxia-inducible factor-1α (HIF-1α) expression is regulated by O2 level. In normal O2 conditions, VHL binds HIF-1α and allows HIF-1α proteasomal degradation. A single-nucleotide polymorphism (SNP) has been found located in the oxygen-dependent degradation domain at codon 582 (C1772T, rs11549465, Pro582Ser). In hypoxia, VHL/HIF-1α interaction is abolished and HIF-1α activates target genes in the nucleus. This study analyzes the impact of genetic alterations and protein expression of VHL and the C1772T SNP of HIF-1α gene (HIF-1α) on prognosis in early-stage ccRCC (pT1a, pT1b, and pT2). Mutational analysis of the entire VHL sequence and the genotyping of HIF-1α C1772T SNP were performed together with VHL promoter methylation analysis and loss of heterozygosis (LOH) analysis at (3p25) locus. Data obtained were correlated with VHL and HIF-1α protein expression and with tumor-specific survival (TSS). VHL mutations, methylation status, and LOH were detected in 51, 11, and 12% of cases, respectively. Our results support the association between biallelic alterations and/or VHL silencing with a worse TSS. Moreover, we found a significant association between the HIF-1α C1772C genotype and a worse TSS. The same association was found when testing the presence of HIF-1α protein in the nucleus. Our results highlight the role of VHL/HIF-1α pathway in RCC and support the molecular heterogeneity of early-stage ccRCC. More important, we show the involvement of HIF-1α C1772T SNP in ccRCC progression.

Cigarette smoke metabolically promotes cancer, via autophagy and premature aging in the host stromal microenvironment.

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.

The KIT Exon 11 Stop Codon Mutation in Gastrointestinal Stromal Tumors: What Is the Clinical Meaning?

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.

Creating a tumor-resistant microenvironment: cell-mediated delivery of TNFα completely prevents breast cancer tumor formation in vivo.

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-ß, 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.

A molecular computational model improves the preoperative diagnosis of thyroid nodules.

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.