Quantitative Analysis of the Expression of Human N-myristoyltransferase 1 (hnmt-1) in Cancers

Human N-myristoyltransferase 1 (hNMT-1) catalyzes the covalent attachment of myristic acid to N-terminal glycine residues (myristoylation) of numerous protein substrates. Overexpression of hNMT-1 in colorectal and gallblad-der cancers makes it a potential biomarker and drug design target for such cancers. In this study, we investigated hNMT-1 expression during the progression of eight different human cancers using quantitative RTPCR. The study results showed that hNMT-1 was up-regulated in breast, colon, lung and ovarian cancers but not kidney, liver, prostate and thyroid cancers. This suggests a role for hNMT-1 as a biomarker for detection of breast, colon, lung and ovarian cancers. This study also suggests the available hNMT-1 inhibitors may be potential therapeutic agents against breast and lung cancers through all disease stages, although their use would likely be limited to early stage colon and ovarian cancers.


INTRODUCTION
Protein modification is an important component of genetic regulation.The structural alterations resulting from protein modification may regulate the biological activity, sub-cellular localization, stability, and turnover of proteins [1][2][3][4][5].Unlike the common modifications (phosphorylation, nucleotidylation and glycosylation), myristoylation is a relatively rare type of acylation.Myristoylation involves the covalent attachment of myristic acid (C14:0) to the Nterminal glycine residue of protein substrates via an amidic linkage [6][7][8].Co-or post-translational myristoylation increases protein lipophilicity and facilitates the protein's association with cellular membranes and/or other hydrophobic protein domains [9,10].
Protein myristoylation is catalyzed by a family of highly conserved enzymes, N-myristoyltransferases (E.C. 2. 1.3.93).These enzymes are ubiquitously distributed in eukaryotic cells [6][7][8][9][10].Previous biochemical and structural studies revealed that N-myristoyltransferases belong to the GCN5related N-acetyltransferase superfamily and possess a pseudo 2-fold symmetry in their catalytic domains [11][12][13].The Nterminal half is more conserved in amino acid residues and is responsible for the binding of myristoyl-coenzyme A (myristoyl-CoA).The C-terminal half has fewer conserved amino acid residues and is responsible for protein substrate recognition.N-myristoyltransferases follow an ordered bi-bi sequential catalytic mechanism (Fig. 1).Myristoyl-CoA binds to the enzymes first, followed by binding of the protein substrate to form a ternary complex.Upon completion of the reaction, coenzyme A (Co-A) is released prior to release of the myristoylated protein substrate.No covalent bonds are formed between myristoyl-CoA and the N-myristoyltransferases during the reaction [14].Human N-myristoyltransferase 1 (hNMT-1) is encoded by a single gene copy located on chromosome 17 (17q21.31)[15,16].hNMT-1 protein substrate specificity can be broadly divided into two groups: signal transduction proteins such as the protein tyrosine kinases pp60 c-src , pp60 yes , pp56 lck , pp59 fyn/syn and c-Abl, and viral proteins such as the HIV-1 and SIV Nef proteins and the HIV Pr53 gag precursor [17][18][19][20][21][22][23][24][25][26][27][28].hNMT-1's involvement in tumorgenesis was first reported with colon cancer where colorectal cancer cells demonstrated enhanced hNMT-1 expression and activity, as well as increased activity of its substrate pp60 c-src [29,30].Furthermore, inhibition of pp60 c-src myristoylation using Nmyristoyltransferase inhibitors depressed the colony formation of colonic cancer cell lines [29].Subsequent reports indicated hNMT-1 upregulation in gallbladder and brain cancers [31,32].Such studies identified hNMT-1 as a possible anti-cancer drug design target, which has promoted the development of several families of inhibitors [33][34][35][36].Since substrates of hNMT-1 participate in different signal transduction pathways, abnormal expression and activity of hNMT-1 might be involved in the pathogenesis of other cancer types.In addition, no previous studies have addressed the

MATERIALS AND METHODOLOGY
hNMT-1 mRNA expression levels were evaluated in an Origene 96-sample TissueScan Oncology qPCR Cancer Survey Panel (Table 1) by quantitative RT-PCR using an Applied Biosystems 7300 Real-Time PCR System (Foster City, California, USA).hNMT-1 expression was normalized to the internal control, -actin in the different patients.Primer sequences (forward: GATGATGACAACATGTTCCGAT-TTGA-TT; reverse: GCCGGAGAGCCCACAAA) and the TaqMan probe (FAM-ATTCCCCGGAGTTTCT) for gene NMT1 (GenBank accession number: AF043324) encoding hNMT-1 were designed and synthesized by Applied Biosystems based on the ITS region.The TaqMan probe was labeled with FAM at 5'-end and non-fluorescent quencher at 3'-end.The quantitative RT-PCR reaction mixture consisted of TaqMan Gene Expression Master Mix (Applied Biosystems), 0.9 μM of each primer for gene NMT1 and the gene encoding -actin, and 0.9 μM of the TaqMan probe.30μL of PCR reaction mixture was added to each well of the 96smaple qPCR Cancer Survey Panel.The amplification was carried out under the following conditions: 2 min at 50°C, 10 min at 95°C, 60 cycles of 15 s at 95°C, and finally 1 min at 60°C.The expression level of hNMT-1 was averaged in each disease stage and normalized to -actin.The fold-difference in mRNA expression at each disease stage was determined by comparison to expression levels in normal patients (stage 0, expression level set as 1).Unpaired t-test with Welch's correction between the hNMT-1 expression levels in normal and cancer patients for each cancer type was performed with GraphPad Prism 4.0 (GraphPad Software, San Diego, California, USA) when possible, with = 0.05.

RESULTS AND DISCUSSION
To evaluate whether hNMT-1 could be used as a biomarker for human cancers, the mRNA expression level of hNMT-1 was quantitatively analyzed in eight different cancer types.As shown in Table 2, hNMT-1 mRNA expression levels were significantly elevated by an average of 3.7, 3.1, 2.3 and 1.8 fold in breast, colon, lung and ovarian cancers, respectively, (P=0.032,P=0.001, P=0.003 and P=0.012 for breast, colon, lung and ovarian cancer, respectively) but not in kidney, liver, prostate and thyroid cancers.These data implicate a role for hNMT-1 as a biomarker for early detection of human breast, colon, lung and ovarian cancers and suggest that hNMT-1 inhibitors could be used as potential therapeutic agents against such cancers.
To obtain a preliminary impression on whether the expression of hNMT-1 varies with cancer progression, we also wished to compare mRNA expression levels at each cancer stage (Fig. 2).However, the small sample size associated with the panel precluded a complete analysis, one that must await a larger scale screening study.Nonetheless, we have gleaned valuable information from our limited analysis, which will provide an important reference for the design of future screenings.In breast and lung cancers, we noted that hNMT-1 expression generally increases with disease progression, such that by stage IV hNMT-1 expression was 4.5and 3.5-fold higher than normal subjects for breast and lung cancer, respectively.These data suggest hNMT-1 may contribute to the development and progression of breast and lung cancer, and further identified potential role of hNMT-1 inhibitors as effective therapeutics against breast and lung cancer throughout disease progression.
In colon cancer, upregulation of hNMT-1 was greatest in early stage cancer (~6.5-fold higher in stage I) (only one 93year old patient; larger scale screening needed) relative to late stage cancer (~3.5-fold increase in stage IV).These data suggest use of hNMT-1 inhibitors as potential therapeutic agents against colon cancer, particularly during early stage progression.This conclusion is consistent with the previous studies that showed N-myristoyltransferase inhibitors depressed the colony formation of colonic cancer cell lines [29].In ovarian cancer, hNMT-1 expression demonstrated a general increase with disease progression until stage IIIa (only one patient; larger scale screening needed) and returned to normal expression levels by stage IV.Although the reason for this return to normal expression levels in late stage ovarian cancer is not known, the data does indicate that hNMT-1 inhibitors might be effective for treatment of early stage ovarian cancer.Furthermore, as a biomarker, early detection of hNMT-1 expression might be necessary for effective hNMT-1 inhibitor based therapy.Interestingly, we noticed a 2-fold decrease and a 3-fold increase in the hNMT-1 expression at stage IV liver cancer and stage III thyroid cancer, respectively, without changes at other stages of diseases' progression.The panel was based on two stage IV liver cancer and two stage-III thyroid cancer patient samples, and this observation could simply represent a spurious result due to the low sample number.Further studies are required to clarify this outcome.

CONCLUSION
In this study, we showed that hNMT-1 expression was up-regulated in human breast, colon, lung and ovarian cancers, which suggests its use as a potential biomarker for these cancers.Furthermore, the available hNMT-1 inhibitors may be potential therapeutic agents against breast and lung cancers through all stages of cancer progression, while their

Fig. ( 1 ).
Fig. (1).Ordered bi-bi sequential catalytic mechanism for Nmyristoyltransferases. Myristoyl-CoA binds prior to the protein substrate and the release of CoA occurs before the release of the myristoylated protein substrate.

Table 2 . The Average Fold Difference (FD) in hNMT-1 mRNA Expression Levels in Patients with Cancer Relative to Patients Without Cancer
use would likely be limited to early stage colon and ovarian cancers.