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Nm23 expression in endometrial and cervical cancer: inverse correlation with lymph node involvement and myometrial invasion.

The expression of nm23 has been shown to correlate in some solid tumours with their metastatic potential and to be associated with a favourable prognosis in human breast cancer and melanoma. In breast and ovarian cancer nm23 expression is also correlated with lymph node involvement. We analysed the expression of nm23-H1 and -H2 in normal endometrium and in endometrial and cervical cancer by both Northern and Western blotting. Cellular localisation of Nm23-H1 was visualised by immunohistochemistry mostly in the cytoplasm. Both isoforms of Nm23 were present in all the samples analysed, and a clear direct correlation between Nm23-H1 and -H2 levels was evident. Median nm23-H2 levels were higher than than -H1 levels in both tissues. Cervical cancer patients with lymph node involvement were shown to have significantly lower protein levels of Nm23 (P < 0.007 for H1 and P < 0.009 for H2), and a similar trend was also evident in endometrial cancer. Furthermore, the degree of myometrial invasion in endometrial cancer patients was also inversely correlated with Nm23-H1 levels of expression (P < 0.003). Nm23 level may therefore be taken into consideration as a new marker in the prognostic characterisation and in the treatment planning of uterine tumour patients. ImagesFigure 1Figure 2Figure 3

Although conflicting results have been obtained in different solid tumours, nm23 is usually classified as a putative metastasis-suppressor gene. Originally isolated by differential hybridisation between two related murine melanoma cell lines distinguished by their metastatic potential (Steeg et al., 1988), Nm23-H1 has been identified with the A subunit of erythrocyte nucleoside diphosphate kinase (NDPK) (Gilles et al., 1991). A second human gene, nm23-H1, was later identified whose product bears 88% amino acid identity with Nm23-H1 and codes for the B subunit of NDPK (Stahl et al., 1991). Cloning of a third gene, designated nm23-H3b has been reported recently (Jiang et al., 1994), indicating that we may be in the presence of a multigene family and of a complex regulatory mechanism.
Despite the high degree of homology between HI and H2, the two proteins differ in structure and function. H2 contains a putative leucine zipper motif (Stahl et al., 1991) and has been identified with both the c-myc transcription factor PuF (Postel et al., 1993) and with the I-factor (differentiation inhibiting factor) purified from mouse myeloid leukaemia cells (Okabe-Kado et al., 1992) and is clearly a bifunctional molecule whose NDPK activity is not required for DNA binding, nor for in vitro transcriptional activity (Postel and Ferrone, 1994).
It is well established that nm23-H1 and more questionably -H2 are involved in tumour metastasis control. A decrease in nm23-HI RNA was observed in highly metastatic tumour cells from various rodent systems (Steeg et al., 1988) and transfection of the H cDNA in murine melanoma and human breast cancer cells was shown to cause a reduction of cell migration in response to serum and defined growth factors and a reduction of tumour metastatic potential 'in vivo' (Leone et al., , 1993a. The biochemical mechanisms by which nm23 acts in metastatic processes have not been clarified yet. NDPK activity of nm23 has been reported as unrelated to its metastasis-suppressor effects , while other biochemical functions of nm23 have been described (Nickerson and Wells, 1984;Ohtsuki et al., 1986;Walton and Gill, 1975;Lombardi et al., 1995;Howlett et al., 1994). Autophosphorylation of serine 44 seems to be directly associated with suppression of metastatic potential in nm23-Hi-transfected murine melanoma cells (MacDonald et al., 1993).
A favourable clinical significance of nm23 overexpression has been reported in breast (Bevilacqua et al., 1989), hepatocellular (Nakayama et al., 1992), prostate (Konishi et al., 1993), and gastric cancer (Nakayama et al., 1993) and in melanoma (Florenes et al., 1992). However, metastatic spread and progression have been correlated with overexpression in colon carcinoma (Myeroff and Markowitz, 1993) and neuroblastoma (Leone et al., 1993b). Therefore, it has been hypothesised that nm23 may play a tissue-specific role and that different regulatory mechanisms may act in different tumours. Moreover, an inverse correlation between nm23 levels and lymph node involvement in breast cancer has been shown for HI and not for H2, thus suggesting a different role for the two genes (Tokunaga et al., 1993).
The role of nm23 in gynaecological tumours has not been extensively investigated yet. It has been reported that ovarian cancer patients with metastatic lymph node involvement have lower nm23 levels than lymph node-negative cases (Mandai et al., 1994;Scambia et al., 1996). Moreover, we demonstrated that the overexpression of nm23-H 1 is associated with survival advantage and greater likelihood of response to chemotherapy in patients with advanced ovarian cancer (Scambia et al., 1996). A recent study reported the absence of nm23 mutations in endometrial cancer (Ambros et al., 1994) but did not address the clinicopathological correlation of mRNA/protein levels.
In the present study nm23-H1 and nm23-H2 levels were analysed both by Northern and Western blotting in two different series of endometrial and cervical tumour samples to determine their possible association with clinicopathological parameters. Materials and methods Patients and sample collection This study was conducted on a total of 13 normal endometria, 45 endometrial and 40 cervical cancer samples taken from patients admitted to the Department of Gynaecology of the Catholic University of Rome. Normal endometrial samples were as follows: three post-menopausal, five in the proliferative and five in the secretory phase of the cell cycle.
All cancer patients were staged according to the FIGO classification (Petterson, 1988) and their tumours were graded as well (GI), moderately (G2) or poorly (G3) differentiated (World Health Organization, 1979).
Myometrial invasion was classified as Ml when tumour infiltration was <50% of myometrial thickness or M2 when it exceeded 50%. Patients were treated by abdominal hysterectomy plus bilateral salpingo-oophorectomy. The cervical cancer patients received cisplatin-based neoadjuvant chemotherapy followed by radical surgery (Benedetti-Panici et al., 1991) or conventional exclusive radiotherapy.
Tissues were frozen in liquid nitrogen immediately after surgery and stored at -80°C until processed. The presence of uterine cervical tumour and endometrial tumour was confirmed by histopathological analysis.
Immunohistochemical analysis Immunohistochemical analysis of Nm23-H1 was performed on three cervical and three endometrial cancers with the mouse anti-human nm23/NDP kinase-A monoclonal antibody (Ylem, Avezzano, Italy) and the avidin-biotin-peroxidase complex detection method (Ylem). At the time of surgery tumours were dissected and fixed for 24 h in neutral buffered formalin. Following fixation blocks were paraffin embedded. Sections (5 gm) were dewaxed in xylene, rehydrated in descending concentrations of alcohol down to 80%, washed in water and treated with 0.3% hydrogen peroxide in methanol for 5 min to remove endogenous peroxidase activity. The sections were then washed in Trisbuffered saline pH 7.6 (TBS) and incubated with normal serum as the blocking reagent to minimise non-specific binding. A 1:100 dilution of the specific anti-human Nm23 MAb was applied for 1 h at room temperature. Normal mouse IgG (Sigma) was used as the negative control. The sections were then incubated with the biotinylated goat antimouse IgG and with avidin-biotin-peroxidase complex for 20 min at room temperature. Finally, the sections were washed in TBS, stained by incubation with amino-9ethylcarbazole in N, N'-dimethylformamide in acetate buffer plus 0.01% hydrogen peroxide for 20 min and counterstained with haematoxylin.

Probes
The specific 3' untranslated regions for nm23-H1 and nm23-H2 were labelled with 32P by random priming (Amersham random priming labelling kit) as described earlier (Stahl et al., 1991). Specific activity was 2.1 + 0.4 x 109 d.p.m. jug-' for nm23-HIA and 1.8+0.2 x 109 for nm23-H2. A human f-actin full-length cDNA probe was used as internal control to confirm the amount of RNA loaded in each lane. RNA samples and Northern blotting analysis Total RNA was obtained by the method of Chomczynsky et al. (1987). Total RNA (20 ug) was separated on 1.2% agarose gels in 2.2 M formaldehyde, capillary-transferred to nylon filters (Duralon, Stratagene, La Jolla, CA, USA) and UV-crosslinked. Duplicate filters were made for hybridisation with the specific nm23-HI and -H2 probes.
Hybridisation was performed at 42°C for 18 h in 50% formamide, 5 x saline sodium citrate (SSC), 1 x Denhardt's solution, 0.2% sodium dodecylsulphate (SDS) and 200 pig ml-' denaturated salmon sperm DNA, with 1 x 106d.p.m./ml-' of the specific probe (Sambrook et al., 1989). After washing, blots were exposed to 3 M XDA plus Trimax films at -80°C with intensifying screens for 6 days. The blots were subsequently rehybridised with 0.5 x 106 d.p.m./ml-IP-actin probe and exposed to radiographic film for 24 h. Nm23 relative intensity was measured by densitometry on an LKB XL laser densitometer (Pharmacia LKB Biothecnology, Uppsala, Sweden) and the numbers obtained were normalised to the corresponding /1actin values.
Preparation of the tissue lysates and Western blotting analysis Frozen tumour tissues were pulverised and homogenised as described earlier (Scambia et al., 1993). The protein concentration was determined using the Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA, USA).
Aliquots of 50 Mg of each protein sample were separated onto a 15% SDS-polyacrylamide gel and electroblotted onto a nitrocellulose membrane (Bio-Rad Laboratories), (Ausubel et al., 1994). After electroblotting the membranes were incubated with 6% non-fat dry milk in 1 x TBST (0.1 M Trizma base, 0.15 M sodium chloride, 0.05% Tween 20, pH 7.4) for blocking and then with a 1:300 dilution of rabbit polyclonal anti-Nm23 antibody (a gift from Dr P S Steeg, NCI, Bethesda, MD, USA) in 3% non-fat dry milk in 1 x TBST. Following incubation with a 1: 2000 dilution with an alkaline phosphatase-conjugated goat anti-rabbit antibody, visualisation of the bound antibody was performed with 5-bromo-4-chloro-3-indolyl-phosphate (BCIP, Sigma) and nitroblue tetrazolium [NBT, Sigma, (Knecht and Dimond, 1984)]. Duplicate gels were run for all the samples and stained with Coomassie blue for control. Samples showing degradation bands were eliminated from the series.
Densitometric quantitation of the intensity of the bands corresponding to Nm23-H1 and -H2 was performed on an LKB XL laser densitometer as described above.

Statistical analysis
Pearson's correlation test was used to analyse the relationship between Nm23-H1 and Nm23-H2 levels of expression and the Mann-Whitney non-parametric test was used to analyse the relationship between Nm23 and clinicopathological characteristics of the patients and the distribution of oestrogen receptor (ER), progesterone receptor (PR), epidermal growth factor receptor (EGFR) and p21 levels.

Results
Immunohistochemical localisation of Nm23 in human endometrial and cervical cancer nm23-H2 levels were significantly higher than -H1 (P <0.003 in endometrial and P < 0.002 in cervical cancer).
Thirteen normal, 21 tumour endometrial samples and 21 tumour cervical samples were analysed by Western blotting using an anti-Nm23 polyclonal antibody. Two bands of 18.5 and 17 kDa corresponding to Nm23-H1 and Nm23-H2 respectively, were visible in all samples as shown in Figure  3 in a representative blot. The measured densitometric values are shown in Table I Figure 4b).
Nm23 and clinicopathological characteristics The distribution of Nm23 proteins according to clinicopathological characteristics is shown in No correlation was shown with stage or grade of differentiation (Table II). Also, no significant correlation was found between Nm23 levels and ER, PR, EGFR and ras-p21 levels (data not shown).
product (Liotta and Steeg, 1990), which displays NDPK activity and is involved in normal cell differentiation and development.

Discussion
The present study is the first report which analyses the expression of nm23-H1 and -H2 in normal and neoplastic uterine tissues both at the mRNA and protein levels. Nm23 was present in all the samples analysed, including normal endometrium suggesting that nm23 is likely to play the role of a 'housekeeping' gene involved in the normal cell functions. This is consistent with the finding that Nm23 is highly conserved in evolution and bears up to 75% identity with the Drosophila abnormal wing discs (AWD) gene   (Scambia et al., 1996), we found no differences in nm23 levels between normal and carcinomatosous endometrium. This finding suggests that nm23 does not play a significant role in endometrial tumour carcinogenesis.
Since nm23-H1 has been reported as the gene which is more directly correlated with the metastatic potential, most of the data which have been published so far dealt with HI more than H2. No extensive analysis has been published to date on the relative distribution of the two genes and although H2 has been identified with the PuF transcription factor (Postel et al., 1993) and with the I-factor (Okabe-Kado et al., 1992), its role remains unclear.
In our series of samples the level of H2 was, on average, significantly higher than HI, at both mRNA and protein level. In particular the Western blots performed with polyclonal antibodies, which recognise both isoforms as two bands of different molecular weights, make it possible to measure the relative intensity of HI and H2 at the same time in the same sample, allowing us to make a direct comparison. These results differ from the data obtained in ovarian tissues (Scambia et al., 1996), thus indicating the possibility that H2 may play a more important role in endometrium and cervix than in other tissues of the female genital tract. H2 has been reported as a very abundant message in breast cancer as well (Tokunaga et al., 1993). A clear direct correlation was observed between the levels of expression of HI and H2. A direct correlation has been reported earlier in ovarian and colon cancer (Mandai et al., 1994;Myeroff and Markowitz, 1993;Scambia et al., 1996), but has not been observed in other tissues. In breast cancer, for example, HI and H2 levels are not correlated with each other and only HI is related to prognosis (Tokunaga et al., 1993). This suggests that different regulatory pathways, which control the absolute and relative levels of HI and H2, may act in different tissues.
At present the clinical significance of nm23 in different malignancies has been poorly investigated. As far as gynaecological tumours are concerned, we have demonstrated that nm23-H1 expression has an independent favourable prognostic role in ovarian cancer (Scambia et al., 1996). The possibility that nm23 expression may have a favourable significance in uterine tumours is also supported by the data reported here. In our series, patients with high levels of nm23 showed a lower incidence of lymph node involvement as had already been reported in breast (Bevilacqua et al., 1989) and ovarian cancer (Viel et al., 1995). Moreover, a high level of nm23 is also correlated with a lesser degree of myometrial invasion.
In conclusion, our data suggest that nm23 may play a role in the biology of endometrial and cervical cancer and support the undertaking of more extensive studies to evaluate whether nm23 may be considered an important marker for defining aggressiveness/metastatic potential of uterine tumours.