Metastasis suppressor function of NM23‐H1 requires its 3′‐5′ exonuclease activity

Abstract

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...

Targeting Tumor Metastasis by Regulating Nm23 Gene Expression

“…The 3'-5' exonuclease are critical for maintenance of genomic stability through DNA repair, replication and recombination (Zhang et al, 2011). The loss of Nm23-H1 expression and its cognate 3'-5' exonuclease activity may be conceive to promote genomic instability and malignant progression.…”

“…The 3'-5' exonucleases and NDPK activities of Nm23-H1 is required for metastasis suppressor function. Zhang and his co-workers reported that human melanoma cell lines, 1205LU which metastasizes to the lungs with high penetrance in rodent experimental model of metastasis is deficient in the expression of both Nm23-H1 and Nm23-H2 protein isofoms (Zhang et al, 2011). The wild type Nm23-H1 inhibits motility and invasion capacity and also all mutant animals exhibited normal motility and invasion suppressing activity indicating that none of the activities (NDPK, hisK, and 3'-5' exonuclease) involves to these phenotype in the 1205LU melanoma cell lines.…”

Progress on Nme (NDP kinase/Nm23/Awd) gene family-related functions derived from animal model systems: studies on development, cardiovascular disease, and cancer metastasis exemplified

“…As discussed by Kaetzel et al 2014, Nme1 apparently increases genetic stability in melanoma, possibly by a 3′-5′ exonuclease activity as well as NTP fueling to DNA polymerases. In line with this interpretation, Nme1 promoted the repair of UV-induced DNA damage to limit melanoma formation (Jarrett et al 2011;Yang et al 2009;Zhang et al 2011).…”

Nucleoside diphosphate kinases (NDPKs) in animal development

“…These multiple functions assigned to the NM23 family of proteins have thus far been difficult to reconcile because the exact cellular function of NM23 has remained unclear. However, beyond nucleoside diphosphate kinase activity several other molecular activities have been linked to NDPKs, such as histidine-dependent protein kinase (histidine phosphotransferase) [18,19] and nuclease activity [20,21], as well as lipid bilayer binding [22][23][24][25][26].…”