Despite 35 years of clinical trials, there is little improvement in one-year survival rates for patients with metastatic melanoma, and the disease is essentially untreatable if not cured surgically. The paucity of chemotherapeutic agents that are effective for treating metastatic melanoma indicates a dire need to develop new therapies. Here, we found a previously unrecognized role for c-Abl and Arg in melanoma progression. We demonstrate that the kinase activities of c-Abl and Arg (c-Abl, Arg) are elevated in primary melanomas (60%), in a subset of benign nevi (33%), and in some human melanoma cell lines. Using siRNA and pharmacological approaches, we show that c-Abl/Arg activation is functionally relevant because it is required for melanoma cell proliferation, survival, and invasion. Significantly, we identify the mechanism by which activated c-Abl promotes melanoma invasion by showing that it transcriptionally upregulates MMP-1, and using rescue approaches we demonstrate that c-Abl promotes invasion via a STAT3→MMP-1 pathway. Additionally, we show that c-Abl and Arg are not merely redundant, as active Arg drives invasion in a STAT3-independent manner, and upregulates MMP-3 and MT1-MMP, in addition to MMP-1. Most importantly, c-Abl and Arg not only promote in vitro processes important for melanoma progression, but also promote metastasis in vivo, as inhibition of c-Abl/Arg kinase activity with the c-Abl/Arg inhibitor, nilotinib, dramatically inhibits metastasis in a mouse model. Taken together, these data identify c-Abl and Arg as critical, novel, drug targets in metastatic melanoma, and indicate that nilotinib may useful in preventing metastasis in patients with melanomas harboring active c-Abl and Arg.

NM23-H1 belongs to a family of eight gene products in humans that have been implicated in cellular differentiation and development, as well as oncogenesis and tumor metastasis. We have defined NM23-H1 biochemically as a 3-5 exonuclease by virtue of its ability in stoichiometric amounts to excise single nucleotides in a stepwise manner from the 3 terminus of DNA. The activity is dependent upon the presence of Mg 2؉ , is most pronounced with single-stranded substrates or mismatched bases at the 3 terminus of double-stranded substrates, and is inhibited by both ATP and the incorporation of cordycepin, a 2-deoxyadenosine analogue, into the 3-terminal position. The 3-5 exonuclease activity was assigned to NM23-H1 by virtue of: 1) precise coelution of enzymatic activity with wild-type and mutant forms of NM23-H1 protein during purification by hydroxylapatite and gel filtration column high performance liquid chromatography and 2) significantly diminished activity exhibited by purified recombinant mutant forms of the proteins. Lysine 12 appears to play an important role in the catalytic mechanism, as evidenced by the significant reduction in 3-5 exonuclease activity resulting from a Lys 12 to glutamine substitution within the protein. 3-5 Exonucleases are believed to play an important role in DNA repair, a logical candidate function underlying the putative antimetastatic and oncogenic activities of NM23-H1.nm23-H1 was first classified as a metastasis suppressor gene on the basis of its reduced expression in several metastatic melanoma cell lines relative to nonmetastatic counterparts (1). Subsequently, low expression of the NM23-H1 protein has been linked to increased metastatic potential in human breast carcinoma (2), hepatoma (3), and gastric carcinoma (4). NM23-H1 overexpression has been shown experimentally to inhibit the metastatic phenotype and/or promote differentiation in melanoma (5), breast carcinoma (6), and transformed neural cell lines (7,8). The human nm23-H1 gene is one of eight related NM23 family members identified to date (reviewed in Ref. 9). Each exhibits nucleoside-diphosphate kinase (NDPK) 1 activity, catalyzing the transfer of ␥-phosphate between nucleoside triphosphate and nucleoside diphosphate via a "ping-pong" mechanism (10). NDPK activity does not appear to be relevant to metastasis suppression, however, as catalytically inactive mutants retain metastasis suppressor activity (11,12). In addition to NDPK, a number of other biological activities have been reported for NM23 proteins, some of which have been proposed to underlie metastasis suppression. Two spontaneous mutations in NM23-H1, a serine 120 to glycine (S120G) substitution seen frequently in aggressive neuroblastomas (13), and a proline 96 to serine mutation that corresponds to the killer of prune mutation (Kpn; P96S) in the Drosophila homologue of NM23 (awd), both result in loss of antimetastatic activity (14). Interestingly, both mutations also abrogate a histidine-dependent protein kinase activity of NM23-H1 (15). This activity has ...

The platelet-derived growth factor (PDGF)-A promoter is regulated by a number of GC-rich regulatory elements that possess non-B-form DNA structures. Screening of a HeLa cDNA expression library with the C-rich strand of a PDGF-A silencer sequence (5-S1 nuclease-hypersensitive site (SHS)) yielded three cDNA clones encoding NM23-H1, a protein implicated as a suppressor of metastasis in melanoma and breast carcinoma. Recombinant human NM23-H1 cleaved within the 3-portions of both 5-SHS strands in either singlestranded or duplex forms. In contrast, NM23-H2, known as a transcriptional activator with a DNA cleavage function, cleaved within the 5-portions of both strands, revealing that NM23-H1 and NM23-H2 cleave at distinct sites of the 5-SHS and by different mechanisms. NM23-H1 and NM23-H2 also cleaved within the PDGF-A basal promoter region, again exhibiting preferences for cleavage within the 5-and 3-portions of the element, respectively. Transient transfection analyses in HepG2 cells revealed that both NM23-H1 and -H2 repressed transcriptional activity driven by the PDGF-A basal promoter (؊82 to ؉8). Activity of the negative regulatory region (؊1853 to ؊883), which contains the 5-SHS, was also inhibited modestly by NM23-H1 and NM23-H2. These studies demonstrate for the first time that NM23-H1 interacts both structurally and functionally with DNA. They also indicate a role for NM23 proteins in repressing transcription of a growth factor oncogene, providing a possible molecular mechanism to explain their metastasis-suppressing effects.The platelet-derived growth factor (PDGF) 1 family consists of three structurally similar glycoproteins (M r 30,000) that induce proliferation and other growth-related effects in cells of mesenchymal origin. These proteins arise from covalent dimerization of two PDGF subunits, designated the A-chain and B-chain, yielding the heterodimer PDGF-AB and two homodimers, PDGF-AA and PDGF-BB (1, 2). PDGF was implicated in tumorigenesis following the discovery of high sequence homology between the PDGF B-chain (PDGF-B) and the viral oncogene, v-sis (for a review, see Ref.3). Other studies suggest that both PDGF-A and PDGF-B may also mediate tumor progression to the metastatic phenotype (4, 5). Transcription of the PDGF-A gene is regulated by several enhancer and silencer elements that are poly-purine/pyrimidine-rich and possess a high degree of single-stranded, non-B DNA structure. Other laboratories (6, 7) as well as our own (8) have demonstrated that a highly GC-rich and nuclease-hypersensitive element (PDGF-A NHE) in the proximal 5Ј-flanking sequence of the PDGF-A promoter (Ϫ82 to Ϫ40) contributes most of the basal transcriptional activity of the gene. This activity is mediated by the binding of members of the Sp1 family of transcription factors and can be induced in vascular endothelial cells by phorbol ester treatment through displacement of Sp1 and Sp3 by the early growth response factor Egr-1 (7, 9) or repressed by binding of the Wilms' tumor gene product WT1 (10). More recently, we local...

ErbB2+ human breast cancer is a major clinical problem. Prior results have suggested that tetraspanin CD151 might contribute to ErbB2-driven breast cancer growth, survival, and metastasis. In other cancer types, CD151 sometimes supports tumor growth and metastasis. However, a definitive test of CD151 effects on de novo breast cancer initiation, growth, and metastasis has not previously been done. We used CD151 gene-deleted mice expressing the MMTV-ErbB2 transgene to show that CD151 strongly supports ErbB2+ mammary tumor initiation and metastasis. Delayed tumor onset (by 70-100 days) in the absence of CD151 was accompanied by reduced survival of mammary epithelial cells and impaired activation of FAK- and MAPK-dependent pathways. Both primary tumors and metastatic nodules showed smooth, regular borders, consistent with a less invasive phenotype. Furthermore, consistent with impaired oncogenesis and decreased metastasis, CD151-targeted MCF-10A/ErbB2 cells showed substantial decreases in three-dimensional colony formation, EGF-stimulated tumor cell motility, invasion, and transendothelial migration. These CD151-dependent functions were largely mediated through α6β4 integrin. Moreover, CD151 ablation substantially prevented PKC- and EGFR/ERK-dependent α6β4 integrin phosphorylation, consistent with retention of epithelial cell polarity and intermediate filament cytoskeletal connections, which helps to explain diminished metastasis. Finally, clinical data analyses revealed a strong correlation between CD151 and ErbB2 expression and metastasis-free survival of breast cancer patients. In conclusion, we provide strong evidence that CD151 collaborates with LB integrins (particularly α6β4 and ErbB2 (and EGFR) receptors to regulate multiple signaling pathways, thereby driving mammary tumor onset, survival, and metastasis. Consequently, CD151 is a useful therapeutic target in malignant ErbB2+ breast cancer.

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...

NM23-H2/NDP kinase B has been identified as a sequence-specific DNA-binding protein with affinity for a nuclease-hypersensitive element of the c-MYC gene promoter (Postel et al., 1993). The ability of Nm23-H2 to activate c-MYC transcription in vitro and in vivo via the same element demonstrates the biological significance of this interaction. Mutational analyses have identified Arg34, Asn69 and Lys135 as critical for DNA binding, but not required for the NDP kinase reaction. However, the catalytically important His118 residue is dispensible for sequence-specific DNA binding, suggesting that sequence-specific DNA recognition and phosphoryl transfer are independent properties. Nm23-H2 also has an activity that cleaves DNA site-specifically, involving a covalent protein-DNA complex. In a DNA sequence-dependent manner, Nm23-H2 recognizes additional target genes for activation, including myeloperoxidase, CD11b, and CCR5, all involved in myeloid-specific differentiation. Moreover, both NM23-H1 and Nm23-H2 bind to nuclease hypersensitive elements in the platelet-derived growth factor PDGF-A gene promoter sequence-specifically, correlating with either positive or negative transcriptional regulation. These data support a model in which NM23/NDP kinase modulates gene expression through DNA binding and subsequent structural transactions.

The NME family of proteins is composed of 10 isoforms, designated NME1-10, which are diverse in their enzymatic activities and patterns of subcellular localization. Each contains a conserved domain associated with a nucleoside diphosphate kinase (NDPK) function, although not all are catalytically active. Several of the NME isoforms (NME1, NME5, NME7, and NME8) also exhibit a 3′–5′ exonuclease activity, suggesting roles in DNA proofreading and repair. NME1 and NME2 have been shown to translocate to the nucleus, although they lack a canonical nuclear localization signal. Binding of NME1 and NME2 to DNA does not appear to be sequence-specific in a strict sense, but instead is directed to single-stranded regions and/or other non-B-form structures. NME1 and NME2 have been identified as potential canonical transcription factors that regulate gene transcription through their DNA-binding activities. Indeed, the NME1 and NME2 isoforms have been shown to regulate gene expression programs in a number of cellular settings, and this regulatory function has been proposed to underlie their well-recognized ability to suppress the metastatic phenotype of cancer cells. Moreover, NME1 and, more recently, NME3, have been implicated in repair of both single- and double-stranded breaks in DNA. This suggests that reduced expression of NME proteins could contribute to the genomic instability that drives cancer progression. Clearly, a better understanding of the nuclear functions of NME1 and possibly other NME isoforms could provide critical insights into mechanisms underlying malignant progression in cancer. Indeed, clinical data indicate that the subcellular localization of NME1 may be an important prognostic marker in some cancers. This review summarizes putative functions of nuclear NME proteins in DNA binding, transcription, and DNA damage repair, and highlights their possible roles in cancer progression.

Human ovarian cancer is diagnosed in the late, metastatic stages but the underlying mechanisms remain poorly understood. We report a surprising functional link between CD151-α3β1 integrin complexes and the malignancy of serous-type ovarian cancer. Analyses of clinical specimens indicate that CD151 expression is significantly reduced or diminished in 90% of metastatic lesions, while it remains detectable in 58% of primary tumors. These observations suggest a putative tumor-suppressing role of CD151 in ovarian cancer. Indeed, our analyses show that knocking down CD151 or α3 integrin enhances tumor cell proliferation, growth and ascites production in nude mice. These changes are accompanied by impaired cell-cell contacts and aberrant expression of E-cadherin, Mucin 5AC and fibronectin, largely reminiscent of an epithelial to mesenchymal transition (EMT)-like change. Importantly, Slug, a master regulator of EMT, is markedly elevated. Knocking down Slug partially restores CD151-α3β1 integrin complex-dependent suppression of cell proliferation. Moreover, disruption of these adhesion protein complexes is accompanied by a concomitant activation of canonical Wnt signaling, including elevated levels of β-catenin and Axin-2 as well as resistance to the inhibition in β-catenin-dependent transcriptional complexes. Together, our study demonstrates that CD151-α3β1 integrin complexes regulate ovarian tumor growth by repressing Slug-mediated EMT and Wnt signaling.