| Abstract|| |
Background: Non-metastatic nm23H1 gene is thought to play a critical role in cell proliferation. Studies of nm23H1 have been done in many other malignancies. But none of these studies took up nm23H1 gene as predictor in the metastases of prostatic carcinoma. Aims and Objectives: To study the expression of nm23H1 in prostatic lesion and to correlate nm23H1 expression with presence of metastases, tumour stage, tumour grade and with PSA level serum. Setting and Design: Tertiary hospital based retrospective and prospective study done in a period of one year from thirty patients having prostatic lesion confirmed by biopsy. Material and Methods: Immunohistochemistry for nm23H1 was performed on unstained coated sections of prostatic lesions to study the relation with prostatic lesion and their correlation with age, PSA level, tumour stage, grading. Clinical data was collected from medical records. Statistical Analysis: SPSS Version 15 analysis software was used. The value were presented in number(%) and Mean ± SD. Results: Majority of patients belong to age group 61 to 70yrs.Gleason score >7 were seen in 55% of patients of adenocarcinoma with and without metastasis. The difference in PSA levels between BPH and adenocarcinoma was significant (P < 0.001). IHC expression for nm23H1 gene showed positive findings in all the cases (P = 1). PSA values >20ng/ml showed maximum % mean expression (98.64%) as compared to PSA levels <10 ng/ml (96.91%). Conclusion: IHC expression of nm23H1 is not an effective tool to distinguish among the cases of BPH, adenocarcinoma of prostate with and without metastasis. Hence nm23H1 gene does not behave like an antimetastatic gene in prostatic lesions.
Keywords: Adenocarcinoma, metastases, nm23H1, prostate
|How to cite this article:|
Kumar A, Hatwal D, Batra N, Verma N. Role of nm23H1 in predicting metastases in prostatic carcinoma. Indian J Pathol Microbiol 2018;61:70-5
| Introduction|| |
The most common diagnosed noncutaneous neoplasm in males is prostate cancer. In term of new cases, it is the sixth most common cancer in the world. In India, it is the second most leading site of cancer among males in large cities and epidemiology also showing rapidly increasing trends in all region of India in all population-based cancer registries., The clinicopathological factor such as Gleason score, tumor stage along with prostate-specific antigen (PSA) level is taken as diagnostic and prognostic factors, these however fails to predict accurate clinical outcome in individual cases., Zinc inhibits nuclear factor-kappa B pathways, antiproliferative and induces apoptosis in abnormal cells., The P13k/Akt signaling cascade works with the transforming growth factor-beta/SMAD signaling cascade to ensure prostate cancer cell survival and protection against apoptosis. Macrophage inhibitory cytokine-1 stimulates the focal adhesion kinase signaling pathway which leads to prostate cancer cell growth and survival. Prostate-specific membrane antigen stimulates the development of prostate cancer by increasing foliate level for the cancer cells to use to survive and growth. Invasion and metastasis of tumor cell provoked by protein that stimulates its attachment to host cellular or extracellular matrix, proteolysis of host barriers to invasion, its locomotion, and colony formation in the organ focused for metastasis., Nonmetastatic nm23 gene plays a critical role to regulate dissemination of tumor cell at molecular level by their ability to inhibit the formation of metastasis while not affecting the growth of primary in vitro., The first metastasis suppressor gene, nm23, was identified in 1988, and since then, eight suppressor genes have been confirmed., Strong expression of nm23H1 likely represents an early event in the development of carcinoma of the prostate. Both protein, nm23H1, and nm23H2 are localized to the cytoplasm, nucleus, and cell membrane. Tumor grade, clinical stage, and serum PSA have become the cornerstone variables in determining treatment options for patients with prostatic carcinoma. Therefore, the present study comprised of clinicopathological correlation of various markers including tumor stage, PSA level, and expression of nm23H1 in prostatic lesions.
Aims and objectives
- To correlate nm23H1 with presence of metastases, tumor stage, tumor grade, and PSA levels
- To study the expression of nm23H1 in prostate cancer versus benign hyperplasia of prostate.
| Materials and Methods|| |
The present study includes selected thirty biopsy-proven cases of benign prostate hyperplasia (BPH) and prostatic carcinoma obtained from needle biopsy, transurethral resections, and total prostatectomy in the period of 1 year at tertiary level hospitals. All the samples were processed for histopathology, stained with hematoxilin and eosin. Sectioned was reviewed for tumor, node, and metastasis (TNM) staging, grading by Gleason system.
Immunohistochemistry (IHC) for the expression of nm 23H1 was performed in unstained paraffin-embedded tissue section using primary antibody nm23H1 mouse monoclonal antibody and secondary antibody mACH universal HRP-polymer kit. Slides were coated with 3-aminopropyl triethoxysilane. Sectiones were deparaffinized and incubated for 30 min in 0.3% hydrogen peroxide in methanol, rinsed with tris-buffered. Antigen retrieval was performed followed by cooling and washing steps in tris-buffer at pH 7.5. Tissue specimen were treated overnight with primary antibody at 4°C followed by 1 h incubation at room temperature and then incubated with secondary antibody. Diluted diaminobenzidine tetrahydrochloride was used to develop color. The preparation was counter stained with hematoxylin. The estimated proportion of intense staining cells was determined in five fields at ×400 and graded as 0 (0%–25%), +1 (26%–50%), +2 (51%–75%), and +3 (76%–100%).
Preoperative serum PSA was performed in all cases (>20 ng/ml were considered as raised PSA level).
The statistical analysis was done using SPSS version 15.0 Statistical Analysis Software. Manufactured by IBM, Chicago, Illinois, USA. The values were represented in number (%) and mean ± standard deviation. Level of significance was expressed as “P”.
| Results|| |
The present study was carried out to study the expression of nm23H1 gene among cases of adenocarcinoma of the prostate with or without metastases and to evaluate its role as a predictor in the metastases of prostatic carcinoma.
The mean age of the patients with adenocarcinoma was 66.90 ± 6.89 years, whereas the same of the patients with BPH was 63.60 ± 7.72 years. On comparing the data statistically, no significant difference was observed between two groups (P = 0.935) [Figure 1].
All the 10 (100%) cases of BPH had PSA levels ≤10 ng/ml. None of cases of BPH had PSA levels between 11 and 20 ng/ml. However, 6 (30%) of adenocarcinoma cases had PSA levels between 11 and 20 ng/ml and remaining 13 (65%) of adenocarcinoma cases had PSA levels above 20 ng/ml. Statistically, the difference in PSA levels of two diagnoses was significant (P< 0.001) [Figure 2].
|Figure 2: Distribution of participants according to prostate-specific antigen level|
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Out of 30 cases enrolled for the study, there were 20 cases of prostate adenocarcinoma (66.7%) and 10 cases were of BPH (33.3%). Out of the 20 cases of adenocarcinoma prostate, a total of 4 (20%) cases had metastatic activity. Remaining 16 (80%) cases did not have a metastatic activity.
The Gleason scores ranged from 5 to 8. Mean Gleason score among patients of adenocarcinoma was 6.55 ± 0.95. There were 55% patients having Gleason score ≥7. Out of 4 cases with metastasis, 3 (75%) had Gleason scores ≥7.
Majority (80%) patients were Stage II adenocarcinoma and only 4 (20%) were Stage IV adenocarcinoma. Out of 4 cases with metastasis, 1 (25%) was diagnosed by biopsy while the remaining 3 (75%) were diagnosed by bone scintigraphy.
IHC expression for nm23H1 gene showed positive findings in all the cases in adenocarcinoma and BPH groups (P = 1) [Figure 3].
|Figure 3: Distribution of participants according to immunohistochemical expression for nm23H1 gene|
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Comparison of mean % expression of gene nm23H1 in patients of adenocarcinoma without metastasis, patients of adenocarcinoma with metastasis, and those with BPH revealed the values were similar. Statistically, there was no significant difference among different diagnostic entities (P = 0.173) [Table 1].
|Table 1: Mean percentage expression of nm23H1 gene in patients of adenocarcinoma with and without metastasis and patients of benign prostate hyperplasia|
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Although as compared to those having Gleason score of lower order (<7), the mean % expression was higher among those having Gleason score of higher order (≥7), the difference was not significant statistically (P< 0.180). No significant association between TNM stage and % mean IHC expression was observed (P > 0.05) [Table 2].
|Table 2: Association of mean percentage nm23H1 expression with Gleason scores and tumor, node, and metastasis staging|
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In all the cases, the grade of expression was 3+. There was no difference in grade of expression in different groups (P = 1); majority of cases in all the groups had strong intensity of expression. Diffuse type was more common in adenocarcinoma with and without metastasis as compared to cases of BPH. However, the association was not significant statistically (P = 0.475) [Table 3] and [Figure 4]a, [Figure 4]b, [Figure 5]a, [Figure 5]b, [Figure 6]a,[Figure 6]b,[Figure 6]c.
|Figure 4: Photomicrograph showing diffuse and strong immunohistochemistry expression of nm23H1 in a case of benign prostate hyperplasia (a) ×2.5 (b) ×5|
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|Figure 5: Photomicrograph showing strong immunohistochemistry expression of nm23H1 in adenocarcinoma of prostate (a) ×10 (b) ×10|
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|Figure 6: Photomicrograph showing diffuse and strong immunohistochemistry expression of nm23H1 in metastatic adenocarcinoma of prostate (a-c) ×10|
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PSA values >20 ng/ml showed maximum % mean expression (98.64%) as compared to PSA levels <10 ng/ml (96.91%); however, the association was statistically nonsignificant (P > 0.05) [Table 4].
|Table 4: Cross-tabulation between nm23H1 expression and prostate-specific antigen levels|
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Receiver operator curve analysis shows that the area under a curve was 0.707. The cutoff ≥96.90% indicates a sensitivity of 100% and a specificity of 46.2%.
| Discussion|| |
The pathogenesis of metastasis consists of linked, sequential, and selective steps that are regulated by transient or permanent changes in different genes. nm23H1/nucleoside-diphosphate kinase is associated with tumor metastasis. Its expression is divergent in various malignant tumors. The mechanism by which nm23H1 exerts its metastasis suppressor functions involves multiple pathway, which remains to be clarified. Yang et al. in their study found inverse correlation of nm23H1 with lymph node metastasis and negative correlation between the expression of nm23H1, VEGF-C mRNA, and microlymphatic count. Lapek et al. in their study discussed the evidence of histamine and aspartic acid phosphorylation in prostate cancer and the potential function of nm23H1 in suppressing the metastasis. In prostate cancer, an inverse relationship between nm23H1 expression and metastatic was described, but a correlation with the progression of primary tumors is controversially discussed., Prowatke et al. found no correlation with clinicopathological parameters of primary prostate carcinoma. Ding et al. observed positive expression of nm23H1 mRNA in prostate cancer and negative correlation between the nm23H1 expression and metastasis., Sethi and Kang and Yadav et al. in their study found that all the metastatic suppressor genes do not influence metastatic potential in similar ways., Lombardi et al. stated that the nm23H1 gene and its protein are expressed physiologically during cell growth and differentiation and that this expression varies in tissues.
In our study, there is no significant difference among all three groups of BPH, adenocarcinoma of the prostate, and adenocarcinoma of the prostate with metastasis in mean percentages of IHC expression, IHC grading, IHC intensity, and diffuse or focal positivity. Our study is in accordance with Igawa et al. stated that in the prostate, nm23H1 does not behave as a metastases suppressor gene, and in the contrary, this is more correlated to metastases. Kandil et al. in their study found that in the prostate, nm23H1 does not behave as metastatic suppressor gene, and in the contrary, it is more correlated to metastasis.
In our study, mean IHC expression was 96.29% in Stage II and 94.50% in Stage IV cases of prostatic carcinoma. Our study shows no significant association between tumor stage and percentage mean of IHC expression. In the present study, higher and lower Gleason grade scores both show almost strong and >94.0% IHC expression and were not statistically significant. However, Stravodimos and Constantinides and Ding et al. concluded that there was negative correlation between nm23H1 staining, tumor stage, and Gleason score.
In the present study, mean PSA levels of patients of adenocarcinoma with and without metastasis were 53.47 ± 98.98 ng/ml as against 5.48 ± 2.72 ng/ml among patients of BPH and were statistically significant (P ≤ 0.001). Hence, our study is consistent with higher level of PSA and adenocarcinoma of the prostate with and without metastasis.
PSA levels are grouped in the <10, 11–20, and >20 ng/ml showing mean percentage of nm23H1 expression 96.91, 97.35, and 98.64, respectively (P > 0.05). Cherry et al. also found large fluctuation of the serum PSA level. Hence, increased level can be found in patients with either localized or metastatic cancer. However, the recent research suggests that the rate of increase of PSA (the PSA velocity) is not a specific marker for prostate cancer.
Majority of patients of BPH and adenocarcinoma with or without metastasis belong to the age range of 61–70 years. No significant difference was observed between two groups.
Metastasis is most common cause of mortality in cancer patients. In the present scenario, further study for targeted molecular therapeutics aimed at tumor cells is required for treating as well preventing metastasis.
Gossypol could have the antimetastatic chemotherapeutic benefit for prostate cancer. It plays a role in metastases suppression, and its mechanism of action in this cell line model may involve downregulation of Bcl-2 and Bcl-xL protein and upregulation of nm23H1 protein.,
| Conclusion|| |
The receiver operating characteristic curve shows nm23H1 expression cannot be used as an effective tool to differentiate among the cases of BPH and adenocarcinoma of the prostate with or without metastases. The present study stated that nm23H1 gene does not behave as metastatic suppressor gene in prostate carcinoma. Hence, the search for other markers of antimetastatic potential in prostate carcinoma should be continued. Various other factors inherent to host and malignancy can be associated with the mechanism that suppresses the metastatic process in this disease.
We are grateful to Professor Suresh Babu and Professor Nuzat Husain for their consistent guidance.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Jemal A, Siegel R, Ward E, Murray T, Xu J, Thun MJ, et al.
Cancer statistics, 2007. CA Cancer J Clin 2007;57:43-66.
Parkin DM. Global cancer statistics in the year 2000. Lancet Oncol 2001;2:533-43.
Jain S, Saxena S, Kumar A. Epidemiology of prostate cancer in India. Meta Gene 2014;2:596-605.
Hariharan K, Padmanabha V. Demography and disease characteristics of prostate cancer in India. Indian J Urol 2016;32:103-8.
] [Full text]
Andriole GL, Crawford ED, Grubb RL 3rd
, Buys SS, Chia D, Church TR, et al.
Mortality results from a randomized prostate-cancer screening trial. N
Engl J Med 2009;360:1310-9.
Goh LK, Liem N, Vijayaraghavan A, Chen G, Lim PL, Tay KJ, et al.
Diagnostic and prognostic utility of a DNA hypermethylated gene signature in prostate cancer. PLoS One 2014;9:e91666.
Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000;100:57-70.
You DJ, Park CR, Lee HB, Moon MJ, Kang JH, Lee C, et al.
Asplicing variant of NME1 negatively regulates NF-κB signaling and inhibits cancer metastasis by interacting with IKKβ. J Biol Chem 2014;289:17709-20.
Leav I, Plescia J, Goel HL, Li J, Jiang Z, Cohen RJ, et al.
Cytoprotective mitochondrial chaperone TRAP-1 as a novel molecular target in localized and metastatic prostate cancer. Am J Pathol 2010;176:393-401.
Watanabe S, Miyata Y, Kanda S, Iwata T, Hayashi T, Kanetake H, et al.
Expression of X-linked inhibitor of apoptosis protein in human prostate cancer specimens with and without neo-adjuvant hormonal therapy. J Cancer Res Clin Oncol 2010;136:787-93.
Narizhneva NV, Tararova ND, Ryabokon P, Shyshynova I, Prokvolit A, Komarov PG, et al.
Small molecule screening reveals a transcription-independent pro-survival function of androgen receptor in castration-resistant prostate cancer. Cell Cycle 2009;8:4155-67.
Boissan M, De Wever O, Lizarraga F, Wendum D, Poincloux R, Chignard N, et al.
Implication of metastasis suppressor NM23-H1 in maintaining adherens junctions and limiting the invasive potential of human cancer cells. Cancer Res 2010;70:7710-22.
Kauffman EC, Robinson VL, Stadler WM, Sokoloff MH, Rinker-Schaeffer CW. Metastasis suppression: The evolving role of metastasis suppressor genes for regulating cancer cell growth at the secondary site. J Urol 2003;169:1122-33.
Lee JH, Marshall JC, Steeg PS, Horak CE. Altered gene and protein expression by nm23-H1 in metastasis suppression. Mol Cell Biochem 2009;329:141-8.
Marino N, Marshall JC, Collins JW, Zhou M, Qian Y, Veenstra T, et al.
Nm23-h1 binds to gelsolin and inactivates its actin-severing capacity to promote tumor cell motility and metastasis. Cancer Res 2013;73:5949-62.
Steeg PS, Bevilacqua G, Kopper L, Thorgeirsson UP, Talmadge JE, Liotta LA, et al.
Evidence for a novel gene associated with low tumor metastatic potential. J Natl Cancer Inst 1988;80:200-4.
Steeg PS. Metastasis suppressors alter the signal transduction of cancer cells. Nat Rev Cancer 2003;3:55-63.
Tong Y, Yung LY, Wong YH. Metastasis suppressors nm23H1 and nm23H2 differentially regulate neoplastic transformation and tumorigenesis. Cancer Lett 2015;361:207-17.
Marino N, Nakayama J, Collins JW, Steeg PS. Insights into the biology and prevention of tumor metastasis provided by the Nm23 metastasis suppressor gene. Cancer Metastasis Rev 2012;31:593-603.
Yang ZS, Xu YF, Huang FF, Ding GF. Associations of nm23H1, VEGF-C, and VEGF-3 receptor in human prostate cancer. Molecules 2014;19:6851-62.
Lapek JD Jr. Tombline G, Kellersberger KA, Friedman MR, Friedman AE. Evidence of histidine and aspartic acid phosphorylation in human prostate cancer cells. Naunyn Schmiedebergs Arch Pharmacol 2015;388:161-73.
Konishi N, Nakaoka S, Tsuzuki T, Matsumoto K, Kitahori Y, Hiasa Y, et al.
Expression of nm23-H1 and nm23-H2 proteins in prostate carcinoma. Jpn J Cancer Res 1993;84:1050-4.
Igawa M, Urakami S, Shiina H, Ishibe T, Usui T, Chodak GW, et al.
Association of nm23 protein levels in human prostates with proliferating cell nuclear antigen expression at autopsy. Eur Urol 1996;30:383-7.
Prowatke I, Devens F, Benner A, Gröne EF, Mertens D, Gröne HJ, et al.
Expression analysis of imbalanced genes in prostate carcinoma using tissue microarrays. Br J Cancer 2007;96:82-8.
Ding GF, Li JC, Xu YF. Study on the correlationship between the expression of nm23H1mRNA, TGF-beta1mRNA and tumor metastases, survival rate with prostate cancer. Fen Zi Xi Bao Sheng Wu Xue Bao 2006;39:544-52.
Andolfo I, De Martino D, Liguori L, Petrosino G, Troncone G, Tata N, et al.
Correlation of NM23-H1 cytoplasmic expression with metastatic stage in human prostate cancer tissue. Naunyn Schmiedebergs Arch Pharmacol 2011;384:489-98.
Sethi N, Kang Y. Unravelling the complexity of metastasis – Molecular understanding and targeted therapies. Nat Rev Cancer 2011;11:735-48.
Yadav VK, Kumar A, Mann A, Aggarwal S, Kumar M, Roy SD, et al.
Engineered reversal of drug resistance in cancer cells – Metastases suppressor factors as change agents. Nucleic Acids Res 2014;42:764-73.
Lombardi D, Lacombe ML, Paggi MG. Nm23: Unraveling its biological function in cell differentiation. J Cell Physiol 2000;182:144-9.
Igawa M, Rukstalis DB, Tanabe T, Chodak GW. High levels of nm23 expression are related to cell proliferation in human prostate cancer. Cancer Res 1994;54:1313-8.
Kandil MA, Moshira M, El-Wahed A. The significance of metastasis related factors nm23H1 and cathepsin D in prostate cancer. J Egyp Nat Cancer Inst 2000;12:199-210.
Stravodimos K, Constantinides C. Manousakas: Immunohistochemical expression of transforming growth factor bet-1 & nm23H1 antioncogene in prostate cancer: Divergent correlation with clinicopathological parameters. Anticancer Res 2000;20:3823-8.
Koo KC, Park SU, Kim KH, Rha KH, Hong SJ, Yang SC, et al.
Predictors of survival in prostate cancer patients with bone metastasis and extremely high prostate-specific antigen levels. Prostate Int 2015;3:10-5.
Cherry JP, Mordente JA, Chapman JR, Choudhury MS, Tazaki H, Mallouh C, et al.
Analysis of cathepsin D forms and their clinical implications in human prostate cancer. J Urol 1998;160:2223-8.
Carter HB. Assessing risk: Does this patient have prostate cancer? J Natl Cancer Inst 2006;98:506-7.
Banerjee S, Jha HC, Robertson ES. Regulation of the metastasis suppressor nm23-H1 by tumor viruses. Naunyn Schmiedebergs Arch Pharmacol 2015;388:207-24.
Huang YW, Wang LS, Dowd MK, Wan PJ, Lin YC. (-)-gossypol reduces invasiveness in metastatic prostate cancer cells. Anticancer Res 2009;29:2179-88.
Carotenuto M, de Antonellis P, Chiarolla CM, Attanasio C, Damiani V, Boffa I, et al.
Atherapeutic approach to treat prostate cancer by targeting Nm23-H1/h-prune interaction. Naunyn Schmiedebergs Arch Pharmacol 2015;388:257-69.
C/o Dr. S.K. Hatwal, Health Care Center, Upper Bazar, Srinagar, Garhwal, Uttarakhand
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2], [Table 3], [Table 4]