Immunolocalisation of c-Myc an Oncoprotein of Canine Mammary Tumors

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Immunolocalisation of c-Myc an Oncoprotein of Canine Mammary Tumors
Gavhane, D.S.,* Singh, A., Sood, N.K., Gupta, K.,Baig, M.R. and Mugale, M.
Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, India.
Email: mmaulivet@gmail.com
* Corresponding author: Dr. Gavhane Dnyaneshwar Shivaji Ph. D. Scholar, Bombay Veterinary College, Mumbai, India. Phone: +91-9011443802.
AB ST RAC T
The study was undertaken to detect the expression pattern of c-Myc, an important oncoprotein of canine mammary tumors by means of immunohistochemistry. A total 31 canine mammary tumors, 2 simple carcinomas, 14 complex carcinomas, 13 carcinosarcomas and 2 sarcomas were included in the study. Immunoreactivity of c-Myc was observed in 87% of cases. Benign lesions showed nuclear localization of c-Myc while it was found to be cytoplasmic in anaplastic malignant cancer cells. c-Myc expression was more prominent in benign and carcinoma cases while it was absent or almost undetectable in sarcomas. These findings suggest that c-Myc is highly expressed in canine mammary tumors and in carcinoma cases may indicate a poor prognosis. c-Myc appears to be involved in tumor progression and may serve as an important target for anti-cancer drugs. Key Words: c-Myc, mammary tumor, carcinoma, carcinosarcoma, sarcoma, immunoreactivity, anti-cancer drug.
MYC, the cellular homolog of v-myc is a well known proto-oncogene and the proteins it express c-Myc, L-myc and N-myc play different roles in cell cycle regulation (1). Among these different proteins the most important and extensively studied is c-Myc a basic helix-loop-helix leucine zipper transcription factor, which appears to be involved in embryogenesis, cell growth, cell differentiation, apoptosis and has transforming potential (2). All these activities are governed by myc–max heterodimer formation in cells and its interaction with DNA E box element. Myc is an early response gene as its response is quickly attained following a variety of mitogenic stimuli (3). The c-Myc expression is undetectable or very low in the resting cell. Under the influence of growth factors or mitogenic stimuli there is rapid increase in the level of c-Myc which arouses the cell from resting phase to enter the G1 phase. This increased level lasts for a short period of time with a
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half life of 20-30 minutes. It is continuously produced during the cell cycle and then declines when cell the cycle is over and the growth factors are withdrawn (4). Normally the expression of c-Myc is low to absent and tightly regulated. The increase in the level of c-Myc induces apoptosis through p53-dependent and independent pathways which result in the release of cytochrome-c from mitochondria, the formation of the Apaf-1/caspase-9, the death effector complex and resultant cell death (5). Various research studies have suggested that aberrant expression of altered or mutant c-Myc prevents the cell from exiting the cell cycle resulting in the accumulation of abnormal undifferentiated precursor cells which are transformed and which ultimately may lead to tumor progression (6). Thus c-Myc may be considered as a potential target for anticancer drugs to impede tumor growth. The role of c-Myc has been studied in various tumors such as breast, cervical, head and neck, prostate, glioma and
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skin cancer (7, 8, 9, 10). Many immunohistochemical studies have shown that about 50–100% of human breast cancer cases have increased levels of c-Myc proteins (11, 12, 13, 14, 15, 16). However there is a dearth of information available about the expression pattern of c-Myc in tumors of animals. This study was therefore carried out to study c-Myc expression in canine mammary tumors, a common tumor in dogs accounting for 25 and 50 percent of all tumors and second in incidence after skin tumors in dogs (17, 18, 19, 20). Furthermore, one third to half of surgically removed canine mammary tumors are malignant (18). MATERIAL AND METHODS
The study was conducted on 31 cases of canine mammary gland tumors presented to the Department of Veterinary Pathology, College of Veterinary Science, GADVASU, Ludhiana, Punjab, India. After preparation of paraffin blocks and histological sections stained with Haematoxylin and Eosin (H&E), stained sections underwent initial screening. Lesions like alveolar hyperplasia, atypical hyperplasia, intraductal papillomas, ductal carcinoma in situ (DCIS), and lobular carcinoma in situ were considered as early lesions, whereas lesions such as invasive ductal carcinoma (IDC), invasive lobular carcinomas and epithelial–mesenchymal transitions (EMT) were considered as advanced lesions (21, 22, 23, 24, 25). EMT in canine mammary tumors were identified based on whether the epithelial structure was preserved or lost, polarity of epithelial cells, and on the basis of immunoreactivity of intermediate filaments like cytokeratin cocktail, vimentin and adhesion molecules like E-cadherin (BioGenex Laboratories Inc., San Ramon, California, USA). Epithelial cells undergoing EMT were positive for both cytokeratin and vimentin protein immunoreactivity, this indicating transition from epithelial to mesenchymal cells. Also cells undergoing EMT showed loss of immunoreactivity to intermediate protein E-cadherin which confirmed EMT (26, 27). After screening, diagnosis and classification of the mammary tumors, immunohistochemistry (IHC) was performed to observe c-Myc protein expression. Cells from tumor emboli were also observed for c-Myc protein expression.
xzylene and rehydrated in descending grades of alcohol. The antigen retrieval was carried out in citrate buffer (pH 6.0). Rabbit polyclonal antibody was used against c-Myc (A-14 Rabbit Polyclonal, SC-789, Santa Cruz Biotechnology Inc., Santa Cruz, California, USA). Immunohistochemical staining was performed by using advanced SSTM One-Step Polymer–HRP IHC Detection System (BioGenex Laboratories Inc., San Ramon, California, USA) as per manufacturer’s instructions. c-Myc expression was analyzed according to its distribution and positivity pattern. Immunoreactivity was expressed as the staining index on the basis of percentage (P) and intensity (I) in the cells showing positive reaction as per Balcanto et al. 2004 (28). Intensity scores were assessed as 0, 1, 2 and 3. Intensity score greater than 1 was considered as high. Depending upon the percentage of cells showing c-Myc immunoreactivity, percentage scores was assessed as 1 (1–25%), 2 (26–50%), 3 (51–75%) and 4 (76–100%). Percentage score more than 3 i.e. when the immunoreactive cells were detected in more than 50% of cells present in microscopic field, was considered as high. RESULTS
Immunohistochemistry
For immunohistochemistry 4-5µ thick sections were taken on positively charged slides. Sections were dewaxed in
A total 31 cases of canine mammary tumors were subjected to immunohistochemistry for the c-Myc protein. Among these 31 cases 2 were simple carcinomas, 14 were complex carcinomas, 13 were carcinosarcomas and 2 were sarcomas (Table 1). In our study the pattern of c-Myc immunolocalisation was both nuclear as well as cytoplasmic. 87% of cases showed the c-Myc expression (Figure 1, 2). Out of 31 cases 18 cases presented with more than 50% cells which were positive for c-Myc expression (i.e. percentage score ≥3). Among 27 tumors which were positive for c-Myc, expression of c-Myc was noticed in both the nucleus and the cytoplasm in 21 cases, whereas only cytoplasmic or nuclear staining was observed in 4 and 2 cases respectively. The nuclear localization of c-Myc protein was predominant in early stage lesions. Invading cells, that is, the neoplastic cells spreading across the basement membrane into mammary tissue, showed nuclear exclusion of c-Myc protein. Thus advanced lesions showed c-Myc localization in cytoplasmic compartment. Some tumor emboli were found to be immunoreactive for c-Myc protein. Cells
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Figure 1: Section of canine mammary tumor showing nuclear immunoreactivity of c-Myc oncoprotein in a case of carcinoma. One Step Polymer HRP Detection System, counterstained with Gills hematoxylin. Original magnification -100X.
Figure 2: Section of canine mammary tumor showing predominantly cytoplasmic immunoreactivity of c-Myc oncoprotein in a case of carcinosarcoma. One Step Polymer HRP Detection System, counterstained with Gills hematoxylin. Original magnification -400X.
from epithelium to mesenchymal transitions (EMT) also showed immunoreactivity to c-Myc protein. Other cells from parenchyma like connective tissue and inflammatory cells were negative for c-Myc immunoreactivity.
munoreactivity was more pronounced in simple and complex carcinomas, it suggests that c-Myc expression is predominant in early lesions. Many immunohistochemical studies have shown that about 50–100% of human breast cancer cases have increased levels of c-Myc proteins (11, 12, 13, 14, 15, 16). In a few studies nuclear localization and the predomiDISCUSSION nant cytoplasmic localization of c-Myc proteins have also In the present study increasing nuclear frequency of c-Myc been observed (29). Mazzini et al. 2009 (30) found more protein was seen in lesions like simple carcinoma particuc-Myc expression in invasive, malignant cells where benign larly ductal carcinoma in situ and lobular carcinoma in situ lesion showed less c-Myc expression. Overall results sugand complex carcinoma while it was more cytoplasmic in gested that the c-Myc expression is greater in neoplastic cell carcinosarcomas and it was found to be down regulated as populations than in simple or complex carcinomas. These tumors progressed to the pure sarcomatous type. The results findings along with the findings of other researchers indicate suggests that the c-Myc protein expression is initially nuclear that c-Myc may be important in progression of neoplasms and then later it becomes progressively more cytoplasmic. from benign to invasive (31). Balcanto et al. 2004 (28) also reported the loss of nuclear Interestingly the c-Myc was also expressed in the cells expression from normal to invasive cells with predominant of epithelial to mesenchymal transition (EMT). The assocytoplasmic expression in invasive cells. As the c-Myc imciation of c-Myc with EMT indicates that c-Myc may play an important role in cell Table 1: Immunostaining of c-Myc oncoprotein in canine mammary tumor (N=31) switch from one cell type to another type, Type of tumor Total no. No. of cases showing Immunoreactivity which are reminiscent of the properties of of cases c-Myc expression stem cells and may be indicative that c-Myc Simple Carcinoma 2 2 More Nuclear may be an important cancer stem cell regulaComplex Carcinoma 14 11 More Nuclear tor (9, 32). Localization of c-Myc in tumor Carcinosarcoma 13 12 More cytoplasmic emboli in few cases indicates that c-Myc may Sarcoma 2 2 Cytoplasmic (very also play a role in tumor metastasis but this low expression) requires further study for verification.
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Overall findings of this study indicated by the immunoreactivity to c-Myc protein suggests that there might be a relation between nuclear expression with early tumor cell populations and cytoplasmic expression with the advanced and invasive tumor cell population as well as EMTs. These findings are based of limited number of cases and therefore it is suggested that further studies be carried out to confirm the results presented here. Predominant cytoplasmic expression and nuclear exclusion suggests a poor prognosis as these tumor cells might have acquired invasive and metastatic potential however this requires confirmation using other metastatic markers. The frequent over expression or altered expression of c-Myc in tumors may be interrelated to the initiation and progression of tumors (33, 34). These findings need to be evaluated in larger number of mammary neoplastic cases. The authors propose that these finding may denote a role for c-Myc as a potential target for anti-cancer drugs.
ACKNOWLED GEMENT: The authors are thankful to the Dean, College of Veterinary Science, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab for providing the facilities to pursue this work.
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