| Abstract|| |
Background: Sodium iodide symporter (NIS), a transporter of iodine is essential for thyroid hormone biosynthesis. It also plays a role in the radioiodine treatment of thyroid cancers. NIS mediated radioiodine transport to breast cancers is under active investigation due to its potential therapeutic utility. Cellular localization and quantification using immunohistochemistry may provide clues for its utility in management of carcinoma breast. Materials and Methods: Human NIS (hNIS) expression was therefore assessed by utilizing a rabbit polyclonal antibody raised against a cloned hNIS in different grades of infiltrating duct carcinoma of breast and its metastatic deposits namely in lymph nodes, bone marrow, and endometrium. Further, hNIS expression was compared with prognostic markers namely estrogen receptor (ER) and progesterone receptor (PR). Results: hNIS was positive in 90.6% cases (29/32) and Scarff-Bloom-Richardson grading was done in 25 cases and 23 cases were NIS positive. Among nongraded cases, 2/2 cases of carcinoma in-situ were positive and 4/5 were positive in cases having post therapy residual tumor status. The strong positivity for hNIS was seen irrespective of ER or PR status and of grade of breast carcinoma and correlated well with western blot analysis. In all the three metastatic sites, NIS was positive in the tumor. Conclusion: These findings indicate the utility of immnohistochemistry for NIS as a new potential prognostic marker and may provide guidance for possible radio iodine therapy in breast cancer patients.
Keywords: Breast cancer, estrogen receptor, human sodium iodide symporter, prognostic markers, progesterone receptor
|How to cite this article:|
Tandon A, Shrivastava A, Kumar A, Prayaga AK, Sundaram C, Godbole MM. Sodium iodide symporter, estrogen receptor, and progesterone receptor expression in carcinoma breast - An immunohistochemical analysis. Indian J Pathol Microbiol 2011;54:745-51
|How to cite this URL:|
Tandon A, Shrivastava A, Kumar A, Prayaga AK, Sundaram C, Godbole MM. Sodium iodide symporter, estrogen receptor, and progesterone receptor expression in carcinoma breast - An immunohistochemical analysis. Indian J Pathol Microbiol [serial online] 2011 [cited 2020 Jan 26];54:745-51. Available from: http://www.ijpmonline.org/text.asp?2011/54/4/745/91514
| Introduction|| |
Sodium iodide symporter (NIS) is a glycosylated protein localized primarily at the basolateral surface of thyroid follicular cells and performs iodide transport function.  This transported iodine is utilized for the synthesis of thyroid hormone (TH) which is known to perform variety of physiological functions in the vertebrates.  Besides this, functional expression of NIS also forms the basis of radioiodine therapy for the treatment of majority of thyroid carcinomas.  Extra thyroidal tissues like lactating breast, salivary gland, and gastric mucosa also express NIS.  Utility of NIS expression in these tissue systems remains to be understood, however, iodide concentrated by the lactating breast is used by infants to synthesize their own TH.  In addition to this, more than 80% of clinical samples of human breast carcinoma as well as their metastases to different parts of the body have been shown to functionally over express human NIS (hNIS). ,, The role of NIS in treating breast cancer and its metastases is still in infancy. ,,
Antibodies against different regions of NIS have been developed and are proved to be valuable in studying normal and pathological thyroid and breast. ,,, In this study, we report raising of specific antisera against cytosolic region of hNIS to recognize hNIS in thyroid, thyroid cancer, breast cancer and its metastases and Michigan Cancer Foundation-7 (MCF- 7) breast cancer cell line (MCF-7) using both immunohistochemical method and western blotting.
NIS expression is governed by several factors in thyroid especially by thyroid stimulating hormone (TSH).  Prolactin influences NIS expression in lactational state and breast cancer.  Other local and endocrine factors play an important role in the progression of breast cancer namely estrogen receptor, progesterone receptor, retinoic acid receptors RAR, RXR, and PPAR. ,, These factors may play a role in the regulation of expression of NIS. NIS expression was also compared with presently available prognostic markers namely estrogen receptor (ER) and progesterone receptor (PR) by IHC. ,,
| Materials and Methods|| |
Raising Antisera Against hNIS
Thyroid tissues from Graves' disease patient were collected after thyroidectomy in the department of Endocrine surgery from Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, India. Written consent was obtained from all the patients. RNA was extracted from 1 gm of Grave's thyroid tissue known to express high level of hNIS.  Total RNA was extracted using 1 gm of tissue using the method of Chomczynski and Sacchi.  Total RNA (2 μg) was transcribed into complementary DNA (cDNA) using Thermoscript reverse transcriptase-polymerase chain reaction (RT-PCR) system according to the manufacturer's protocol (Life Technologies, Inc. Calasbad, CA, USA). Two μl of cDNA was used as template for PCR using the primers designed to amplify 1924b to 2279b sequence corresponding to 525-643 amino acids of hNIS (High antigenic sites). Sequence of primers is as follows: Sense primer: GGA TCC GCC ATC TCC TAT CTC TAT TAC GGT and antisense primer: GTC GAG CTC GAG TCA GAG GTT TGT CTC CTG CTG G. This amplified hNIS cDNA fragment corresponding to 354bp is ligated into glutathione-S-transferse (GST) fusion vector pGEX-4T-2 (Amersham Pharmacia Biotech, Buckinghamshire, UK) between the BamHI and XhoI sites. Sequence and orientation was confirmed using Sanger's dideoxy termination DNA sequencing (ABI Prism 373, Applied Biosystems, Foster city CA, USA). The pGEX 4T-2 vector carrying hNIS cDNA was transformed into the E coli-BL21 (DE3) strain and clones positive for hNIS were confirmed by PCR using primers described earlier. Fusion protein expression was induced by adding isopropyl-β-D-thio-galactopyranoside (IPTG) at a final concentration of 1 mM. Fusion protein was purified using Glutathione Sepharose 4B (Amersham Biosciences, Buckinghamshire, UK) and presence of GST-hNIS fusion protein was confirmed by western blotting using antibodies against GST and hNIS both. To raise antibodies, two rabbits were immunized subcutaneously with GST-hNIS fusion protein (200 μg) emulsified with complete Freund's adjuvant. Rabbits were immunized after every four weeks. Ten days after each immunization, rabbits were bled and serum was collected. After 13 weeks, serum was obtained from the rabbits. Specificity of the raised antisera was determined using a monoclonal antibody against hNIS, a kind gift from Dr. J.C. Morris (Mayo clinic, Rochester, USA).
The membrane protein was isolated by homogenizing the breast cancerous tissue and breast cancerous cell line MCF-7 in five volumes of the buffer (10 mM Tris-HCl, pH 7.4, 5 mM NaCl, 1 mM EDTA, 0.1 mM PMSF, and 50 mg/ml leupeptin) containing 0.25 M sucrose. After gentle homogenization at 4°C, the lysates were centrifuged at 700 g for 10 min at 4°C. The supernatant was further centrifuged at 100,000 g for 90 min at 4°C to collect total postnuclear membrane and cytosolic fractions. Both the fractions were resuspended in the homogenizing buffer and stored at -80°C. Purity of cytosolic and membrane fraction was confirmed using biochemical assay for lactate dehydrogenase and cytochrome oxidase, respectively. Samples from the membrane and cytosolic fraction (30 μg) were added to the sample buffer (10% glycerol, 2% SDS, 0.0625 M Tris-HCl, pH 6.8) and heated with 2-mercaptoethanol at 70°C for 5 min. The samples were subjected to 0.1% sodium dodecyl sulfate (SDS)-12% polyacrylamide gel electrophoresis (PAGE) and electro-transferred to nitrocellulose membranes. Nonspecific binding sites on the membrane were blocked with 5% non fat milk for 1 h. The membrane was then incubated for 2 h at room temperature with polyclonal anti rabbit antibody. A previously described mouse monoclonal NIS antibody (10), a kind gift from Dr. John C. Morris, (Mayo Clinic, Rochester, USA) was used as a reference (not shown). After washing three times with tris-buffered saline tween 20 (TBST), the membranes were incubated with appropriate horseradish peroxidase-conjugated secondary antibody for 1 h. The filter was washed three times with TBST. Proteins were detected by Supersignal® west pico chemiluminescent substrate (Pierce, Rockford, IL, USA). NIS overexpressing Grave's tissue was used as a positive control.
NIS protein expression was analyzed in carcinoma breast tissue samples using conventional whole tissue sections (CWTS). The cases were retrieved from records of the pathology department of Nizam's Institute of Medical Sciences, Hyderabad, India. Thirty-two cases were randomly selected, ages ranging from 27 to 67 years with mean age of 52.5 with a single male patient. Thirty cases were of infiltrating duct carcinomas (IDC) and two cases were in-situ ductal carcinomas. Scarff Bloom Richardson grading had been given for all these cases and immunohistochemistry for ER and PR was performed. Immunohistochemical study for NIS antibody was done. NIS Positivity was interpreted membrane and/or cytoplasmic staining. Graves' thyroiditis and peritumoral normal breast tissue were taken as positive control in study [Figure 1]c and d. NIS immunohistochemistry was done in cases with metastasis in infiltrating duct carcinomas (IDC). There were three cases among them one case show lymph node metastasis second case show bone marrow metastasis and third case show uterine metastasis in follow-up.
|Figure 1: (a) Western blot analysis of breast cancer tissue sample: Cell lysate from Membrane fractions from Graves' thyroid (lane 1), breast carcinoma tissue (lane 2,3), and from breast cancer MCF-7 cells. Two bands corresponding to 77 kda and 55 kda hNIS were detected. (b) Intracellular localization of hNIS: Breast cancer membrane fraction (lane 1, 3) and cytosolic fraction (lane 2,4) prepared from tissue samples Presence of two 77 kda and 55 kda forms were detected. (c) Graves' disease tissue thyroid showed membrane and cytoplasmic positivity of moderate to strong intensity, (d) peritumoral and normal breast tissue showed predominantly NIS negative epithelium, (e) Fibrocystic disease of breast showed basolateral, membrane positivity of mild to moderate intensity. (c-e, ×20)|
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In brief, 4-μm sections were mounted on poly-l-lysine coated slides. All sections were deparaffinized at 60°C for 30 min. Slides were washed through three changes of xylene and hydrated through alcohols to distilled water. Antigen retrieval was performed using 10% citrate buffer in a pressure cooker for 40 min and rapid cooling was achieved with distilled water. Tissues were incubated in 3% H 2 O 2 for 15 min to quench endogenous peroxides. All washes were performed with TBST (0.3 M NaCl, 0.1% Tween 20, and 0.05 M Tris-HCl, pH 7.6) three times for 5 min each time. Sections were blocked with serum-free protein and endogenous biotin activity was blocked with Biotin Blocking System (DAKO Corp. CA, USA). Slides were incubated for two hours with primary antibody diluted in serum-free protein block. The streptavidin-biotin method as specified by the supplier was followed (CSA kit, DAKO Corp. CA, USA). Peroxidase activity was detected with diaminobenzidene-hydrogen peroxide and was observed as a brown product. All slides were counterstained with hematoxylin. Similar routine protocols were followed for monoclonal mouse anti-human estrogen receptor α, Clone 1D5, (DAKO Corp. CA, USA) 1:50 dilution and polyclonal rabbit anti-human progesterone receptor, dilution 1:50, (DAKO Corp.CA, USA).
| Results|| |
Western Blotting Results
Mouse monoclonal NIS antibody (10), a kind gift from Dr. John C. Morris, (Mayo Clinic, Rochester, USA), was used as a reference and was compared with raised polyclonal antibody. The Two identical bands corresponding to 77 kda and 55 kda hNIS were detected.
Western blot analysis of breast cancer tissue sample
The cell lysate from membrane fractions from Graves' disease [[Figure 1]a; lane 1], breast carcinoma tissue [[Figure 1]a; lane 2,3], and from breast cancer MCF-7 cells was evaluated. Total 30 μg of lysate was electrophoresed and transferred on nitrocellulose membrane. Two bands corresponding to 77 kda and 55 kda hNIS were detected.
Intracellular localization of hNIS
Breast cancer membrane fraction [[Figure 1]b; lane 1,3] and cytosolic fraction [[Figure 1]b; lane 2,4] prepared from tissue samples. Presence of two 77 kda and 55 kda forms were detected.
Expression of NIS in Human Breast Cancer
Expression of hNIS was examined in breast cancer tissue samples. Two bands corresponding to 77 kda and 55 kda were identified using the technique of western blotting. Grave's thyroid, a pathological condition in which NIS is over expressed, was used as a positive control. NIS protein was found to be expressed in human breast cancer cell line MCF-7. Presence of NIS was confirmed in both cytosolic as well as membrane fraction [Figure 1]a and b.
NIS Expression in Various Stages of Breast Neoplasm
NIS expression in premalignant lesions of breast
We studied NIS expression in single case of fibrocystic disease of breast (FDP) and two cases of carcinoma in-situ. In FDP, NIS expression was observed at the basolateral aspect of plasma membrane [Figure 1]e. In ductal carcinoma in-situ, one case was 80% and another was 90% positive for NIS. Intense positivity was observed both in membrane and cytoplasm [Table 1].
NIS expression in invasive duct carcinoma of breast
Scarff-Bloom-Richardson grading was done in 25 cases of invasive duct carcinoma breast. Among different grades, NIS was positive in 23/25 (92%) cases and expression is irrespective of grading [Table 1]. In all the tissues examined intense positivity was observed both in membrane and cytoplasm [Figure 2]a-f.
|Figure 2: Grading of carcinoma breast and NIS expression (a-f), grade1 (a), grade2 (c); (H and E, ×20)and grade3 (e H and E, ×40) NIS expression in (e, ×10), (e, ×20), (f, ×40); NIS at metastatic sites with tumor (g-l), lymph node (g, h) metastasis show strong expression (g, ×10), lymphoid parenchyma negative (h, ×40), bone marrow biopsy core (l, ×20 and J, ×40) positive in both tumor and native marrow cells including megakaryocytes (arrow), endometrium (k, l, ×20) tumor is strong positive, endometrial glands show variable positivity|
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NIS expression in carcinoma breast with post-therapy status
In this subgroup, five cases were studied. Prior to surgery for residual tumor, two cases received chemotherapy, two cases received both chemotherapy and radiotherapy and one case received only radiotherapy. NIS expression was observed in 4/5 (80%) cases with both membrane and cytoplasmic positivity in the range of 60%-85% tumor cells [Table 1].
NIS expression in metastatic deposits of carcinoma breast to different sites of body
We further assessed NIS expression in breast cancer metastasis. One sample was taken from lymph node showing metastasis. Tumor cells showed strong, membrane and cytoplasmic positivity in 60% tumor cells [Figure 2]g; while native lymphoid stroma is predominantly negative [Figure 2]h. In another case of bone marrow metastasis, NIS showed strong, membrane and cytoplasmic positivity in 85% tumor cells [Figure 2]i. Interestingly megakaryocytes and other myeloid, erythroid precursors of marrow also showed strong, membrane and cytoplasmic positivity for NIS [Figure 2]j. Third case showed metastasis in endometrium. Tumor cells were seen adjacent to normal endometrial glands without cellular or nuclear atypia. NIS showed strong, membrane and cytoplasmic positivity in 80% of metastatic tumor cells [Figure 2]k. Variable staining of normal endometrial glands were observed with few glands showing negative to weak membrane positivity [Figure 2]l and other glands were strong membrane positive [Figure 2]k.
NIS Expression and ER and PR Status
The immunohistochemical correlation of NIS was examined with estrogen receptor and progesterone receptor in all the 32 cases [Table 2]. Eight cases were ER negative and nine were PR negative, out of these, six cases were negative for both ER and PR. Remaining 21 cases showed positivity for both ER and PR, however, a large variation in their percentage positivity was recorded. The intense membrane and cytoplasmic NIS immunoreactivity was observed in 29 out of 32 cases irrespective of ER and PR positivity [Figure 3]a-l. In three cases negative for NIS, positivity for both ER and PR was recorded.
|Figure 3: ER, PR and NIS immunostaining (a-l), index case (a-c, ×40); nuclear positivity (a) for ER, negative nuclei (b) for PR, NIS (c); index case (d-f, ×40); negative nuclei (d) for ER, nuclear positivity (e) for PR, NIS (f); index case (g-i, ×40); nuclear positivity (g) for ER, nuclear positivity (h) for PR, NIS (i); index case (j-l, ×40); negative nuclei (j) for ER, negative nuclei (k) for PR, NIS (l). NIS strong membrane and cytoplasmic signal|
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|Table 2: NIS immunopositivity (tumor cells cytoplasm and membrane score 3)|
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| Discussion|| |
It is confirmed that radioiodine therapy for thyroid carcinoma is mediated through the functional NIS. The possible use of radioiodine for breast cancer treatment is suggested by the presence of NIS. ,,, Since the possibility of radioiodine use is largely dependent on the presence of a functional NIS. In the present work, we performed immunohistochemical analysis of the tissue samples of breast carcinoma for it presence and semi-quantification. We also analyzed the presence of ER and PR in these samples by IHC to check for relative utility of NIS in comparison with these prognostic markers. Further, we looked for the hNIS expression in metastasizing tumors at bone marrow, endometrium, and lymph nodes in three separate patient samples.
NIS expression was checked by IHC using a polyclonal antibody raised against a c-terminus specific hNIS cloned in our laboratory. The ability of the polyclonal antibody to detect the presence of hNIS in cytosolic and membrane fractions was initially checked by western blotting [Figure 1]a and b.
Our results demonstrate intense hNIS immunoreactivity in both membrane and cytoplasm in 65% to 90% of breast tumor cells in 90.2% of samples. In majority of tumor specimen examined, the presence of hNIS was irrespective of either tumor grade or its ER and/or PR status. Its strong presence was also recorded even in carcinoma in-situ as well as in samples from patients undergoing chemotherapy and/or radiotherapy. These results are in agreement with the earlier studies providing evidence that breast carcinomas are amenable to radioiodine therapy as suggested by others. ,, The evidence that only about one fourth of the patients demonstrate a reasonable degree of iodine uptake in NIS positive patients indicates that demonstration of NIS per se may not be an adequate criterion for its therapeutic use. 
The presence of NIS irrespective of degree of ER and PR status as well as tumor grade and also in one third of the samples showing the absence of ER and/or PR suggests that hNIS positivity along with a demonstrable radio iodine uptake may serve as an alternate therapeutic approach in patients after unsuccessful hormone based therapy. It is premature to predict the value of NIS as a prognostic marker if and when such a therapy will come in use. Another study by our group show NIS expression in fibroadenoma of breast  and Ryan et al. demonstrate NIS as potential regulators in normal, benign and malignant human breast tissue, recently.  Scoring system proposed by Wapnir el al. is not relevant since almost all cases are in score 3+ in infiltrating duct carcinoma analyzed by us [Table 1].
Demonstration of hNIS in metastasis to bone marrow and native marrow cells as well as metastasis to endometrium and endometrium glands for the first time underscores the importance of hNIS by us along with its demonstration in metastasis to lymph nodes by others and in the present study indicates a potential use of hNIS immunohistochemsitry. Recently, Renier et al. demonstrated NIS expression in brain metastasis. The demonstration that post therapeutic residual tumors also express hNIS calls to attention the importance of NIS IHC as a tool for providing useful information for their management with radioiodine therapy.
| Acknowledgment|| |
This work was supported by Life Science Research Board, Defense Research and Development Organization (Grant No. DBAS/48222/LSRB-9/2000/BB/RD-81 to Prof. Madan M.Godbole). Ashutosh Shrivastava is a recipient of research fellowship from Council of Scientific and Industrial Research, New Delhi (9/590(36)/2002/EMR-II).
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Madan M Godbole
Department of Endocrinology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Raebareli Road, Lucknow - 226 014
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2]