Molecular skin fluorescence imaging: A tool for evaluating early melanoma development.

A novel approach to melanoma diagnosis-in vivo molecular skin fluorescence imaging (mSFI)-was developed to identify premalignant changes in the form of tissue remodeling related to melanoma development in humans by imaging the proximal microenvironment of lesions. The method was tested using a fluorescent peptide (ORL-1) which binds to αvß3 integrin, a molecule associated with invasive melanoma development. A cut off score of 7 was established, differentiating melanomas from nonmelanoma nevi with 100% sensitivity, and 95.7% specificity, while identifying dysplastic nevi with the potential for melanoma development.

Transcriptome Analysis Identifies Oncogenic Tissue Remodeling during Progression from Common Nevi to Early Melanoma.

Early detection and treatment of melanoma, the most aggressive skin cancer, improves the median 5-year survival rate of patients from 25% to 99%. Melanoma development involves a stepwise process during which genetic changes drive histologic alterations within nevi and surrounding tissue. Herein, a comprehensive analysis of publicly available gene expression data sets of melanoma, common or congenital nevi (CN), and dysplastic nevi (DN), assessed molecular and genetic pathways leading to early melanoma. The results demonstrate several pathways reflective of ongoing local structural tissue remodeling activity likely involved during the transition from benign to early-stage melanoma. These processes include the gene expression of cancer-associated fibroblasts, collagens, extracellular matrix, and integrins, which assist early melanoma development and the immune surveillance that plays a substantial role at this early stage. Furthermore, genes up-regulated in DN were also overexpressed in melanoma tissue, supporting the notion that DN may serve as a transitional phase toward oncogenesis. CN collected from healthy individuals exhibited different gene signatures compared with histologically benign nevi tissue located adjacent to melanoma (adjacent nevi). Finally, the expression profile of microdissected adjacent nevi tissue was more similar to melanoma compared with CN, revealing the melanoma influence on this annexed tissue.

Bayesian gene set analysis for identifying significant biological pathways.

We propose a hierarchical Bayesian model for analyzing gene expression data to identify pathways differentiating between two biological states (e.g., cancer vs. non-cancer and mutant vs. normal). Finding significant pathways can improve our understanding of biological processes. When the biological process of interest is related to a specific disease, eliciting a better understanding of the underlying pathways can lead to designing a more effective treatment. We apply our method to data obtained by interrogating the mutational status of p53 in 50 cancer cell lines (33 mutated and 17 normal). We identify several significant pathways with strong biological connections. We show that our approach provides a natural framework for incorporating prior biological information, and it has the best overall performance in terms of correctly identifying significant pathways compared to several alternative methods.

A pluripotency signature predicts histologic transformation and influences survival in follicular lymphoma patients.

Histologic transformation (HT) of follicular lymphoma to diffuse large B-cell lymphoma (DLBCL-t) is associated with accelerated disease course and drastically worse outcome, yet the underlying mechanisms are poorly understood. We show that a network of gene transcriptional modules underlies HT. Central to the network hierarchy is a signature strikingly enriched for pluripotency-related genes. These genes are typically expressed in embryonic stem cells (ESCs), including MYC and its direct targets. This core ESC-like program was independent of proliferation/cell-cycle and overlapped but was distinct from normal B-cell transcriptional programs. Furthermore, we show that the ESC program is correlated with transcriptional programs maintaining tumor phenotype in transgenic MYC-driven mouse models of lymphoma. Although our approach was to identify HT mechanisms rather than to derive an optimal survival predictor, a model based on ESC/differentiation programs stratified patient outcomes in 2 independent patient cohorts and was predictive of propensity of follicular lymphoma tumors to transform. Transformation was associated with an expression signature combining high expression of ESC transcriptional programs with reduced expression of stromal programs. Together, these findings suggest a central role for an ESC-like signature in the mechanism of HT and provide new clues for potential therapeutic targets.

Electron microscopy localization and characterization of functionalized composite organic-inorganic SERS nanoparticles on leukemia cells.

We demonstrate the use of electron microscopy as a powerful characterization tool to identify and locate antibody-conjugated composite organic-inorganic nanoparticle (COINs) surface enhanced Raman scattering (SERS) nanoparticles on cells. U937 leukemia cells labeled with antibody CD54-conjugated COINs were characterized in their native, hydrated state using wet scanning electron microscopy (SEM) and in their dehydrated state using high-resolution SEM. In both cases, the backscattered electron (BSE) detector was used to detect and identify the silver constituents in COINs due to its high sensitivity to atomic number variations within a specimen. The imaging and analytical capabilities in the SEM were further complemented by higher resolution transmission electron microscopy (TEM) images and scanning Auger electron spectroscopy (AES) data to give reliable and high-resolution information about nanoparticles and their binding to cell surface antigens.

Genomic and proteomic analysis reveals a threshold level of MYC required for tumor maintenance.

MYC overexpression has been implicated in the pathogenesis of most types of human cancers. MYC is likely to contribute to tumorigenesis by its effects on global gene expression. Previously, we have shown that the loss of MYC overexpression is sufficient to reverse tumorigenesis. Here, we show that there is a precise threshold level of MYC expression required for maintaining the tumor phenotype, whereupon there is a switch from a gene expression program of proliferation to a state of proliferative arrest and apoptosis. Oligonucleotide microarray analysis and quantitative PCR were used to identify changes in expression in 3,921 genes, of which 2,348 were down-regulated and 1,573 were up-regulated. Critical changes in gene expression occurred at or near the MYC threshold, including genes implicated in the regulation of the G(1)-S and G(2)-M cell cycle checkpoints and death receptor/apoptosis signaling. Using two-dimensional protein analysis followed by mass spectrometry, phospho-flow fluorescence-activated cell sorting, and antibody arrays, we also identified changes at the protein level that contributed to MYC-dependent tumor regression. Proteins involved in mRNA translation decreased below threshold levels of MYC. Thus, at the MYC threshold, there is a loss of its ability to maintain tumorigenesis, with associated shifts in gene and protein expression that reestablish cell cycle checkpoints, halt protein translation, and promote apoptosis.

Inhibition of HMGcoA reductase by atorvastatin prevents and reverses MYC-induced lymphomagenesis.

Statins are a class of drugs that inhibit 3-hydroxy-3-methylglutaryl-coenzyme A (HMGcoA) reductase, a critical enzyme in the mevalonate pathway. Several reports document that statins may prevent different human cancers. However, whether or not statins can prevent cancer is controversial due to discordant results. One possible explanation for these conflicting conclusions is that only some tumors or specific statins may be effective. Here, we demonstrate in an in vivo transgenic model in which atorvastatin reverses and prevents the onset of MYC-induced lymphomagenesis, but fails to reverse or prevent tumorigenesis in the presence of constitutively activated K-Ras (G12D). Using phosphoprotein fluorescence-activated cell sorter (FACS) analysis, atorvastatin treatment was found to result in the inactivation of the Ras and ERK1/2 signaling pathways associated with the dephosphorylation and inactivation of MYC. Correspondingly, tumors with a constitutively activated K-Ras (G12D) did not exhibit dephosphorylation of ERK1/2 and MYC. Atorvastatin's effects on MYC were specific to the inhibition of HMGcoA reductase, as treatment with mevalonate, the product of HMG-CoA reductase activity, abrogated these effects and inhibited the ability of atorvastatin to reverse or suppress tumorigenesis. Also, RNAi directed at HMGcoA reductase was sufficient to abrogate the neoplastic properties of MYC-induced tumors. Thus, atorvastatin, by inhibiting HMGcoA reductase, induces changes in phosphoprotein signaling that in turn prevent MYC-induced lymphomagenesis.

Rehabilitation of cancer through oncogene inactivation.

The inactivation of the MYC oncogene alone can reverse tumorigenesis. Upon MYC inactivation, tumors stereotypically reverse, undergoing proliferative arrest, cellular differentiation and/or apoptosis. The precise consequences of MYC inactivation appear to depend upon both genetic and epigenetic parameters. In some types of cancer following MYC inactivation, tumor cells become well differentiated and biologically and histologically normal, inducing sustained tumor regression. However, in some cases, these normal-appearing cells are actually dormant tumor cells and upon MYC reactivation they rapidly recover their tumorigenic properties. Future therapies to treat cancer will need to address the possibility that tumor cells can camouflage a normal phenotype following treatment, resting in a dormant, latently cancerous state.

Tumor dormancy and MYC inactivation: pushing cancer to the brink of normalcy.

Upon MYC inactivation, tumors variously undergo proliferative arrest, cellular differentiation, and apoptosis and in some cases, apparently permanently revoking tumorigenesis. In liver tumor cells, we recently showed that MYC inactivation uncovers stem cell properties and triggers differentiation, but in this case, their neoplastic properties are restorable by MYC reactivation. Thus, whereas oncogene inactivation can push cancer to the brink of normalcy, some cells retain the latent capacity to turn cancerous again, arguing that they may exist in a state of tumor dormancy.

MYC inactivation uncovers pluripotent differentiation and tumour dormancy in hepatocellular cancer.

Hepatocellular carcinoma is generally refractory to clinical treatment. Here, we report that inactivation of the MYC oncogene is sufficient to induce sustained regression of invasive liver cancers. MYC inactivation resulted en masse in tumour cells differentiating into hepatocytes and biliary cells forming bile duct structures, and this was associated with rapid loss of expression of the tumour marker alpha-fetoprotein, the increase in expression of liver cell markers cytokeratin 8 and carcinoembryonic antigen, and in some cells the liver stem cell marker cytokeratin 19. Using in vivo bioluminescence imaging we found that many of these tumour cells remained dormant as long as MYC remain inactivated; however, MYC reactivation immediately restored their neoplastic features. Using array comparative genomic hybridization we confirmed that these dormant liver cells and the restored tumour retained the identical molecular signature and hence were clonally derived from the tumour cells. Our results show how oncogene inactivation may reverse tumorigenesis in the most clinically difficult cancers. Oncogene inactivation uncovers the pluripotent capacity of tumours to differentiate into normal cellular lineages and tissue structures, while retaining their latent potential to become cancerous, and hence existing in a state of tumour dormancy.

Conditional animal models: a strategy to define when oncogenes will be effective targets to treat cancer.

The ability to model cancer in the mouse has provided a robust methodology to dissect the molecular etiology of cancer. These models serve as potentially powerful platforms to preclinically evaluate novel therapeutics. In particular, the recent development of strategies to conditionally induce the or knockout the function of genes in a tissue specific manner has enabled investigators to engineer mice to demonstrate that the targeted inactivation of specific oncogenes can be effective in inducing sustained regression of tumors. Thus, these animal models will be useful to define the specific genes that will be therapeutically useful to target for the treatment of particular human cancers.