Marian Novak

Metastasis Suppressor Function of NM23-H1 Requires its 3’–5’ Exonuclease Activity

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′‐5′ exonuclease. Although the histidine kinase has been implicated in suppression of motility in breast carcinoma cell lines, potential relevance of the NDPK and 3′‐5′ 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′‐5′ exonuclease alone (Glu5 to Ala, or E5A), the NDPK and histidine kinase activities tandemly (Y52A, H118F) or all 3 activities simultaneously (K12Q). 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 E5A and K12Q. The H118F mutant, which lacked both the NDPK and histidine kinase while retaining the 3′‐5′ 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′‐5′ 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.

High grade serous ovarian carcinomas originate in the fallopian tube

High-grade serous ovarian carcinoma (HGSOC) is the most frequent type of ovarian cancer and has a poor outcome. It has been proposed that fallopian tube cancers may be precursors of HGSOC but evolutionary evidence for this hypothesis has been limited. Here, we perform whole-exome sequence and copy number analyses of laser capture microdissected fallopian tube lesions (p53 signatures, serous tubal intraepithelial carcinomas (STICs), and fallopian tube carcinomas), ovarian cancers, and metastases from nine patients. The majority of tumor-specific alterations in ovarian cancers were present in STICs, including those affecting TP53, BRCA1, BRCA2 or PTEN. Evolutionary analyses reveal that p53 signatures and STICs are precursors of ovarian carcinoma and identify a window of 7 years between development of a STIC and initiation of ovarian carcinoma, with metastases following rapidly thereafter. Our results provide insights into the etiology of ovarian cancer and have implications for prevention, early detection and therapeutic intervention of this disease.It has previously been proposed that high-grade serous ovarian carcinoma (HGSOC) may originate from the fallopian tube. Here, the authors analyze genetic aberrances in fallopian tube lesions, ovarian cancers, and metastases from HGSOC patients and establish the evolutionary origins of HGSOC in the fallopian tube.

Stathmin 1 and p16INK4A are sensitive adjunct biomarkers for serous tubal intraepithelial carcinoma

OBJECTIVE To credential Stathmin 1 (STMN1) and p16(INK4A) (p16) as adjunct markers for the diagnosis of serous tubal intraepithelial carcinoma (STIC), and to compare STMN1 and p16 expression in p53-positive and p53-negative STIC and invasive high-grade serous carcinoma (HGSC). METHODS Immunohistochemistry (IHC) was used to examine STMN1 and p16 expression in fallopian tube specimens (n=31) containing p53-positive and p53-negative STICs, invasive HGSCs, and morphologically normal FTE (fallopian tube epithelium). STMN1 and p16 expression was scored semiquantitatively by four individuals. The semiquantitative scores were dichotomized, and reported as positive or negative. Pooled siRNA was used to knockdown p53 in a panel of cell lines derived from immortalized FTE and HGSC. RESULTS STMN1 and p16 were expressed in the majority of p53-positive and p53-negative STICs and concomitant invasive HGSCs, but only scattered positive cells were present in morphologically normal FTE. Both proteins were expressed consistently across multiple STICs from the same patient and in concomitant invasive HGSC. Knockdown of p53 in immortalized FTE cells and in four HGSC-derived cell lines expressing different missense p53 mutations did not affect STMN1 protein levels. CONCLUSIONS This study demonstrates that STMN1 and p16 are sensitive and specific adjunct biomarkers that, when used with p53 and Ki-67, improve the diagnostic accuracy of STIC. The addition of STMN1 and p16 helps to compensate for practical limitations of p53 and Ki-67 that complicate the diagnosis in up to one third of STICs.

Mutant p53 regulates ovarian cancer transformed phenotypes through autocrine matrix deposition

High-grade serous ovarian carcinoma (HGS-OvCa) harbors p53 mutations and can originate from the epithelial cell compartment of the fallopian tube fimbriae. From this site, neoplastic cells detach, survive in the peritoneal cavity, and form cellular clusters that intercalate into the mesothelium to form ovarian and peritoneal masses. To examine the contribution of mutant p53 to phenotypic alterations associated with HGS-OvCA, we developed live-cell microscopy assays that recapitulate these early events in cultured fallopian tube nonciliated epithelial (FNE) cells. Expression of stabilizing mutant variants of p53, but not depletion of endogenous wild-type p53, in FNE cells promoted survival and cell-cell aggregation under conditions of cell detachment, leading to the formation of cell clusters with mesothelium-intercalation capacity. Mutant p53R175H-induced phenotypes were dependent on fibronectin production, α5β1 fibronectin receptor engagement, and TWIST1 expression. These results indicate that FNE cells expressing stabilizing p53 mutants acquire anchorage independence and subsequent mesothelial intercalation capacity through a mechanism involving mesenchymal transition and matrix production. These findings provide important new insights into activities of mutant p53 in the cells of origin of HGS-OvCa.

Establishment of patient-derived tumor xenograft models of epithelial ovarian cancer for preclinical evaluation of novel therapeutics

Purpose: Ovarian cancer is the leading cause of death from gynecologic malignancy in the United States, with high rates of recurrence and eventual resistance to cytotoxic chemotherapy. Model systems that allow for accurate and reproducible target discovery and validation are needed to support further drug development in this disease. Experimental Design: Clinically annotated patient-derived xenograft (PDX) models were generated from tumor cells isolated from the ascites or pleural fluid of patients undergoing clinical procedures. Models were characterized by IHC and by molecular analyses. Each PDX was luciferized to allow for reproducible in vivo assessment of intraperitoneal tumor burden by bioluminescence imaging (BLI). Plasma assays for CA125 and human LINE-1 were developed as secondary tests of in vivo disease burden. Results: Fourteen clinically annotated and molecularly characterized luciferized ovarian PDX models were generated. Luciferized PDX models retain fidelity to both the nonluciferized PDX and the original patient tumor, as demonstrated by IHC, array CGH, and targeted and whole-exome sequencing analyses. Models demonstrated diversity in specific genetic alterations and activation of PI3K signaling pathway members. Response of luciferized PDX models to standard-of-care therapy could be reproducibly monitored by BLI or plasma markers. Conclusions: We describe the establishment of a collection of 14 clinically annotated and molecularly characterized luciferized ovarian PDX models in which orthotopic tumor burden in the intraperitoneal space can be followed by standard and reproducible methods. This collection is well suited as a platform for proof-of-concept efficacy and biomarker studies and for validation of novel therapeutic strategies in ovarian cancer. Clin Cancer Res; 23(5); 1263–73. ©2016 AACR.

Potential contributions of antimutator activity to the metastasis suppressor function of NM23-H1

Abstractnm23-h1 is a well-documented metastasis suppressor gene whose mechanism(s) of action have yet to be fully elucidated. The purpose of this report is to discuss recent advances in investigating the potential role of a novel 3′–5′ exonuclease activity identified recently in our laboratory, a biochemical function associated, in general, with DNA repair and replication. We have employed a site-directed mutagenesis approach to demonstrate that the 3′–5′ exonuclease activity of NM23-H1 is required for its metastasis suppressor function. Consistent with a role in DNA repair, we also observe that the single yeast NM23 homolog (YNK1) is required for the maintenance of genomic integrity and normal kinetics of DNA repair in response to exposure to ultraviolet radiation. These results and their implications for understanding the molecular mechanisms underlying NM23-H1 functions in cancer are discussed.

Multiple mechanisms underlie metastasis suppressor function of NM23-H1 in melanoma

Abstractnm23-h1 was the first metastasis suppressor gene to be identified in humans, with early studies demonstrating its ability to inhibit the metastatic potential of breast carcinoma and melanoma cell lines. This report outlines recent findings from our laboratory indicating that the metastasis suppressor function of NM23-H1 in human melanoma involves a spectrum of molecular mechanisms. Analysis of NM23-H1-dependent profiles of gene expression in human melanoma cell lines has identified a host of target genes that appear to mediate suppression of directional motility. Of particular interest is a subset of motility-suppressing genes whose regulation by NM23-H1 is independent of its known kinase and 3′–5′ exonuclease activities. In parallel, we have recently observed that NM23-H1 expression appears to be required for genomic stability and for optimal repair of DNA damage produced by ultraviolet radiation and other agents. Thus, NM23-H1 might oppose not only the motile and invasive characteristics of metastatic cells but also the acquisition of mutations that drive malignant progression to the metastatic phenotype itself.

Metastasis suppressor NME1 regulates melanoma cell morphology, self-adhesion and motility via induction of fibronectin expression

Expression of the metastasis suppressor NME1 in melanoma is associated with reduced cellular motility and invasion in vitro and metastasis in vivo, but the underlying molecular mechanisms are not completely understood. Herein, we report a novel mechanism through which NME1 controls melanoma cell morphology via upregulation of the extracellular matrix (ECM) protein fibronectin. Expression of NME1 strongly suppressed cell motility in melanoma cell lines 1205LU and M14. The resulting sedentary phenotype was associated with a more flattened appearance and marked increases in actin stress fibre and focal adhesion formation. NME1‐induced focal adhesions were colocalized with dense deposits of fibronectin, which were absent or minimal in the corresponding NME1‐deficient parental lines. NME1 was a strong inducer of fibronectin mRNA and protein expression, shown with reciprocal approaches of forced NME1 expression and shRNA‐mediated knock‐down. Increased synthesis and ECM deposition of fibronectin was necessary for NME1‐induced cell spreading, as knock‐down of fibronectin opposed the effects of NME1 on cell morphology. Fibronectin knock‐down also reversed the ability of NME1 to promote aggregation when cells were plated on a non‐adherent substratum. Similarly, inhibiting activation of the fibronectin receptor integrin α4β1 with an anti‐α4 antibody reversed the motility‐suppressing effect of NME1. A positive correlation was observed between NME1 and fibronectin mRNA in clinical biopsies of normal skin, benign nevi and primary melanomas, but not in metastatic forms, suggesting the NME1/fibronectin axis represents a barrier to melanoma progression. In summary, these findings indicate fibronectin is an important effector of the motility‐suppressing function of NME1 in melanoma cells.

Interrogation of Functional Cell-Surface Markers Identifies CD151 Dependency in High-Grade Serous Ovarian Cancer

The degree of genetic aberrations characteristic of high-grade serous ovarian cancer (HGSC) makes identification of the molecular features that drive tumor progression difficult. Here, we perform genome-wide RNAi screens and comprehensive expression analysis of cell-surface markers in a panel of HGSC cell lines to identify genes that are critical to their survival. We report that the tetraspanin CD151 contributes to survival of a subset of HGSC cell lines associated with a ZEB transcriptional program and supports the growth of HGSC tumors. Moreover, we show that high CD151 expression is prognostic of poor clinical outcome. This study reveals cell-surface vulnerabilities associated with HGSC, provides a framework for identifying therapeutic targets, and reports a role for CD151 in HGSC.

Dual functions of NME1 in suppression of cell motility and enhancement of genomic stability in melanoma

The NME1 gene represents the prototypical metastasis suppressor, whose expression inhibits cell motility and metastasis without impact on primary tumor growth in a number of different human cancers. This report outlines our recent efforts to define the molecular mechanisms through which NME1 both suppresses cell motility and promotes genomic integrity in the setting of human melanoma. Forced NME1 expression in a variety of melanoma-derived cell lines was shown to induce dynamic changes in cell morphology and reorganization of the actin cytoskeleton, with formation of a network of thick stress fibers and assembly of fibronectin fibrils at large focal adhesions. Moreover, NME1 expression results in adhesion reprogramming through an impact on integrin repertoire and focal adhesion dynamics. Having previously demonstrated that NME1 expression promotes repair of DNA damage induced by ultraviolet radiation (UVR) in both yeast and mammalian cells, probably via the nucleotide excision repair pathway, we have more recently demonstrated that NME1 is rapidly recruited to double-strand breaks. This preliminary result represents the first evidence of direct interactions between NME1 and DNA in the context of DNA repair and has set the stage for current efforts to probe its functional interactions with double-strand break repair pathways. Discussed herein are molecular models to explain the interactions of NME1 with such diverse cellular functions as cell motility and DNA repair, potentially through its nucleoside diphosphate kinase and 3′-5′ exonuclease activities.