BackgroundCancer staging and treatment presumes a division into localized or metastatic disease. We proposed an intermediate state defined by ≤5 cumulative metastasis(es), termed oligometastases. In contrast to widespread polymetastases, oligometastatic patients may benefit from metastasis-directed local treatments. However, many patients who initially present with oligometastases progress to polymetastases. Predictors of progression could improve patient selection for metastasis-directed therapy.MethodsHere, we identified patterns of microRNA expression of tumor samples from oligometastatic patients treated with high-dose radiotherapy.ResultsPatients who failed to develop polymetastases are characterized by unique prioritized features of a microRNA classifier that includes the microRNA-200 family. We created an oligometastatic-polymetastatic xenograft model in which the patient-derived microRNAs discriminated between the two metastatic outcomes. MicroRNA-200c enhancement in an oligometastatic cell line resulted in polymetastatic progression.ConclusionsThese results demonstrate a biological basis for oligometastases and a potential for using microRNA expression to identify patients most likely to remain oligometastatic after metastasis-directed treatment.

Due to the large number of putative microRNA gene targets predicted by sequence-alignment databases and the relative low accuracy of such predictions which are conducted independently of biological context by design, systematic experimental identification and validation of every functional microRNA target is currently challenging. Consequently, biological studies have yet to identify, on a genome scale, key regulatory networks perturbed by altered microRNA functions in the context of cancer. In this report, we demonstrate for the first time how phenotypic knowledge of inheritable cancer traits and of risk factor loci can be utilized jointly with gene expression analysis to efficiently prioritize deregulated microRNAs for biological characterization. Using this approach we characterize miR-204 as a tumor suppressor microRNA and uncover previously unknown connections between microRNA regulation, network topology, and expression dynamics. Specifically, we validate 18 gene targets of miR-204 that show elevated mRNA expression and are enriched in biological processes associated with tumor progression in squamous cell carcinoma of the head and neck (HNSCC). We further demonstrate the enrichment of bottleneckness, a key molecular network topology, among miR-204 gene targets. Restoration of miR-204 function in HNSCC cell lines inhibits the expression of its functionally related gene targets, leads to the reduced adhesion, migration and invasion in vitro and attenuates experimental lung metastasis in vivo. As importantly, our investigation also provides experimental evidence linking the function of microRNAs that are located in the cancer-associated genomic regions (CAGRs) to the observed predisposition to human cancers. Specifically, we show miR-204 may serve as a tumor suppressor gene at the 9q21.1–22.3 CAGR locus, a well established risk factor locus in head and neck cancers for which tumor suppressor genes have not been identified. This new strategy that integrates expression profiling, genetics and novel computational biology approaches provides for improved efficiency in characterization and modeling of microRNA functions in cancer as compared to the state of art and is applicable to the investigation of microRNA functions in other biological processes and diseases.

Gene expression signatures that are predictive of therapeutic response or prognosis are increasingly useful in clinical care; however, mechanistic (and intuitive) interpretation of expression arrays remains an unmet challenge. Additionally, there is surprisingly little gene overlap among distinct clinically validated expression signatures. These “causality challenges” hinder the adoption of signatures as compared to functionally well-characterized single gene biomarkers. To increase the utility of multi-gene signatures in survival studies, we developed a novel approach to generate “personal mechanism signatures” of molecular pathways and functions from gene expression arrays. FAIME, the Functional Analysis of Individual Microarray Expression, computes mechanism scores using rank-weighted gene expression of an individual sample. By comparing head and neck squamous cell carcinoma (HNSCC) samples with non-tumor control tissues, the precision and recall of deregulated FAIME-derived mechanisms of pathways and molecular functions are comparable to those produced by conventional cohort-wide methods (e.g. GSEA). The overlap of “Oncogenic FAIME Features of HNSCC” (statistically significant and differentially regulated FAIME-derived genesets representing GO functions or KEGG pathways derived from HNSCC tissue) among three distinct HNSCC datasets (pathways:46%, p<0.001) is more significant than the gene overlap (genes:4%). These Oncogenic FAIME Features of HNSCC can accurately discriminate tumors from control tissues in two additional HNSCC datasets (n = 35 and 91, F-accuracy = 100% and 97%, empirical p<0.001, area under the receiver operating characteristic curves = 99% and 92%), and stratify recurrence-free survival in patients from two independent studies (p = 0.0018 and p = 0.032, log-rank). Previous approaches depending on group assignment of individual samples before selecting features or learning a classifier are limited by design to discrete-class prediction. In contrast, FAIME calculates mechanism profiles for individual patients without requiring group assignment in validation sets. FAIME is more amenable for clinical deployment since it translates the gene-level measurements of each given sample into pathways and molecular function profiles that can be applied to analyze continuous phenotypes in clinical outcome studies (e.g. survival time, tumor volume).

Oligometastasis is a clinically distinct subset of metastasis characterized by a limited number of metastases potentially curable with localized therapies. We analyzed pathways targeted by microRNAs over-expressed in clinical oligometastasis samples and identified suppression of cellular adhesion, invasion, and motility pathways in association with the oligometastatic phenotype. We identified miR-127-5p, miR-544a, and miR-655-3p encoded in the 14q32 microRNA cluster as co-regulators of multiple metastatic pathways through repression of shared target genes. These microRNAs suppressed cellular adhesion and invasion and inhibited metastasis development in an animal model of breast cancer lung colonization. Target genes, including TGFBR2 and ROCK2, were key mediators of these effects. Understanding the role of microRNAs expressed in oligometastases may lead to improved identification of and interventions for patients with curable metastatic disease, as well as an improved understanding of the molecular basis of this unique clinical entity.

The metastasis suppressor NM23-H1 possesses 3 enzymatic activities in vitro, a nucleoside diphosphate kinase (NDPK), a protein histidine kinase and a more recently characterized 3 0 -5 0 exonuclease. Although the histidine kinase has been implicated in suppression of motility in breast carcinoma cell lines, potential relevance of the NDPK and 3 0 -5 0 exonuclease to metastasis suppressor function has not been addressed in detail. To this end, site-directed mutagenesis and biochemical analyses of bacterially expressed mutant NM23-H1 proteins have identified mutations that disrupt the 3 0 -5 0 exonuclease alone (Glu 5 to Ala, or E 5 A), the NDPK and histidine kinase activities tandemly (Y 52 A, H 118 F) or all 3 activities simultaneously (K 12 Q). Although forced expression of NM23-H1 potently suppressed spontaneous lung metastasis of subcutaneous tumor explants derived from the human melanoma cell line 1205LU, no significant metastasis suppressor activity was obtained with the exonuclease-deficient variants E 5 A and K 12 Q. The H 118 F mutant, which lacked both the NDPK and histidine kinase while retaining the 3 0 -5 0 exonuclease, also exhibited compromised suppressor activity. In contrast, each mutant retained the ability to suppress motility and invasive characteristics of 1205LU cells in culture, indicating that the NM23-H1 molecule possesses an additional activity(s) mediating these suppressor functions. These studies provide the first demonstration that the 3 0 -5 0 exonuclease activity of NM23-H1 is necessary for metastasis suppressor function and further indicate cooperativity of the 3 enzymatic activities of the molecule on suppression of the metastatic process.Metastasis suppressors are a class of genes defined by their ability to selectively inhibit the metastatic process with little or no impact on primary tumor growth. 1 The first metastasis suppressor gene to be identified was nm23-M1, which was discovered by virtue of its low expression in K-1735-derived melanoma cell lines with elevated metastatic potential. 2 Subsequent studies have confirmed that the human homolog nm23-H1 exhibits potent metastasis suppressor activity in breast carcinoma and melanoma cell lines and is underexpressed in multiple forms of metastatic cancer. 3 Although the mechanisms underlying metastasis suppressor activity of NM23-H1 are not fully understood, the protein exhibits 3 different enzymatic activities in vitro that represent potential antimetastatic functions. First to be described was its nucleoside diphosphate kinase (NDPK) activity, which maintains balance in intracellular nucleotide pools by catalyzing transfer of c-phosphate between nucleoside triphosphates and diphosphates. 4 Although a role for the NDPK in metastasis suppression has been challenged, 5-7 the concept has yet to be addressed directly with in vivo models of metastatic growth. NM23-H1 is also reported to exhibit a protein histidine kinase activity that mediates its antimotility function, 8 possibly via serine phosphorylation of the kinase suppressor...

Reduced expression of the metastasis suppressor NM23-H1 is associated with aggressive forms of multiple cancers. Here, we establish that NM23-H1 (termed H1 isoform in human, M1 in mouse) and two of its attendant enzymatic activities, the 3′-5′ exonuclease and nucleoside diphosphate kinase, are novel participants in the cellular response to UV radiation (UVR)-induced DNA damage. NM23-H1 deficiency compromised the kinetics of repair for total DNA polymerase-blocking lesions and nucleotide excision repair of (6-4) photoproducts in vitro. Kinase activity of NM23-H1 was critical for rapid repair of both polychromatic UVB/UVA (290-400 nm)- and UVC (254 nm)-induced DNA damage, while its 3′-5′ exonuclease activity was dominant in the suppression of UVR-induced mutagenesis. Consistent with its role in DNA repair, NM23-H1 rapidly translocated to sites of UVR-induced (6-4) photoproduct DNA damage in the nucleus. In addition, transgenic mice hemizygous-null for nm23-m1 and nm23-m2 exhibited UVR-induced melanoma and follicular infundibular cyst formation, and tumor-associated melanocytes displayed invasion into adjacent dermis, consistent with loss of invasion-suppressing activity of NM23 in vivo. Taken together, our data demonstrate a critical role for NM23 isoforms in limiting mutagenesis and suppressing UVR-induced melanomagenesis.

Differentiation programs are aberrant in cancer cells allowing them to express differentiation markers in addition to their tissue of origin. In the present study, we demonstrate the multi-lineage differentiation potential of breast cancer cell lines to express multiple neuronal/glial lineage-specific markers as well as mammary epithelial and melanocytic-specific markers. Multilineage expression was detected in luminal (MCF-7 and SKBR3) and basal (MDA-MB-231) types of human breast cancer cell lines. We also observed comparable co-expression of these three cell lineage markers in MDA-MB-435 cells in vitro, in MDA-MB-435 primary tumors derived from parental and single cell clones and in lung metastases in vivo. Furthermore, ectoderm multi-lineage transdifferentiation was also found in human melanoma (Ul-MeL) and glioblastoma cell lines (U87 and D54). These observations indicate that aberrant multi-lineage transdifferentiation or lineage infidelity may be a wide spread phenomenon in cancer.

NM23-H1 is a metastasis suppressor protein that exhibits 3'-5' exonuclease activity in vitro. As 3'-5' exonucleases are generally required for maintenance of genome integrity, this activity represents a plausible candidate mediator of the metastasis suppressor properties of the NM23-H1 molecule. Consistent with an antimutator function, ablation of the yeast NM23 homolog, YNK1, results in increased mutation rates following exposure to UV irradiation and exposure to the DNA damaging agents etoposide, cisplatin, and MMS. In human cells, a DNA repair function is further suggested by increased NM23-H1 expression and nuclear translocation following DNA damage. Also, forced expression of NM23-H1 in NM23-deficient and metastatic cell lines results in coordinate downregulation of multiple DNA repair genes, possibly reflecting genomic instability associated with the NM23-deficient state. To assess the relevance of the 3'-5' exonuclease activity of NM23-H1 to its antimutator and metastasis suppressor functions, a panel of mutants harboring defects in the 3'-5' exonuclease and other enzymatic activities of the molecule (NDPK, histidine kinase) have been expressed by stable transfection in the melanoma cell line, 1205Lu. Pilot in vivo metastasis assays indicate 1205Lu cells are highly responsive to the metastasis suppressor effects of NM23-H1, thus providing a valuable model for measuring the extent to which the nuclease function opposes metastasis and metastatic progression.