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Heat shock proteins HSP90, HSP70 and GRP78 expression in medullary thyroid carcinoma
Annals of Diagnostic Pathology 26 (2017) 52–56
Contents lists available at ScienceDirect
Annals of Diagnostic Pathology
Heat shock proteins HSP90, HSP70 and GRP78 expression in medullary thyroid carcinoma Ethan Soudry a,d,⁎, Sagit Stern Shavit a,d, Britta Hardy c,d, Sarah Morgenstern b,d, Tuvia Hadar a,d, Raphael Feinmesser a,d a
Department of Otolaryngology - Head and Neck Surgery, Beilinson Campus, Petah Tiqwa, Israel Department of Pathology, Rabin Medical Center, Beilinson Campus, Petah Tiqwa, Israel c Felsenstein Research Medical Center, Beilinson Campus, Petah Tiqwa, Israel d Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel b
a r t i c l e
i n f o
Keywords: Medullary thyroid carcinoma Heat shock protein 70 Heat shock protein 90 GRP 78 Immunohistochemistry
a b s t r a c t Background: Medullary thyroid carcinoma management consists mainly of surgical resection and is largely chemoresistant. There is ongoing effort to discover novel therapies for medullary thyroid carcinoma. Increased levels of heat shock proteins have been associated with multiple cancers and are being studied as potential therapeutic targets. The purpose of this study was to determine the expression levels of heat shock proteins 90 and 70 and of glucose related protein 78 in medullary thyroid carcinoma tissues compared with normal thyroid tissues. Methods: 20 tissue specimens of medullary thyroid carcinoma and 10 specimens of thyroids without malignancy were analyzed by immunohistochemistry. Results: Medullary thyroid carcinoma specimens showed 27% higher expression level of heat shock protein 90 immunostaining, and a 43% higher expression level of heat shock protein 70 immunostaining versus normal controls. These differences, however, were not statistically significant. A significantly higher expression level was noted for glucose related protein 78 in the medullary thyroid carcinoma specimens than in the controls. Conclusion: This study indicates increased expression levels of heat shock proteins 90 and 70 and glucose related protein 78 levels in medullary thyroid carcinoma. These findings, though preliminary imply that these proteins may have a role in medullary thyroid carcinoma's tumor biology and may have and future therapeutic options. Larger cohorts are needed to corroborate these results. © 2016 Elsevier Inc. All rights reserved.
1. Introduction Medullary thyroid carcinoma (MTC) is an endocrine neoplasm that arises in the parafollicular C cells of the thyroid gland and may secrete calcitonin and carcinoembryonic antigen. It accounts for 2% to 5% of all thyroid carcinomas [1]. Seventy-five percent of cases are sporadic and the rest are inherited. Up to 50% of sporadic cases and 95% of inherited cases are associated with mutations in the RET proto-oncogene [2]. The clinical course is less aggressive than in anaplastic carcinomas but more aggressive than in well-differentiated thyroid cancers. Regional and distant metastases are frequent (up to 50% of cases). Current management is mainly consisted of surgical resection which includes total thyroidectomy and lymph-node dissection. Radioactive iodine treatment is not effective because, owing to their parafolliccular C-cell origin, MTCs do not take up iodine. External beam radiation therapy has also not been found effective, and there is no effective chemotherapy regimen [1,3]. ⁎ Corresponding author at: Department of Otorhinolaryngology - Head and Neck Surgery, Rabin Medical Center - Beilinson Hospital, Petach Tikva 49100, Israel. E-mail address: [email protected] (E. Soudry).
http://dx.doi.org/10.1016/j.anndiagpath.2016.11.003 1092-9134/© 2016 Elsevier Inc. All rights reserved.
The prognosis of patients with MTC is directly related to disease stage. The overall 10-year survival rate is 61% to 75%, but it decreases to 45% if cervical nodes are involved [3,4]. Therefore, researchers are seeking promising new therapeutic modalities for patients with MTC unresponsive to traditional therapy. Potential targets are thyroid molecular signaling pathways and factors associated with cancer cell biology including vascular endothelial growth factor receptors and antibodies, angiogenesis inhibitors, tyrosine kinase inhibitors, and heat shock protein (HSP) inhibitors [1]. In 1963, HSPs were identified as a unique family of proteins that are expressed following exposure to protein-damaging environmental stresses to promote protein renaturation, restore homeostasis and enhance survival. The process is termed the heat shock response [5,6]. The heat shock family of proteins is divided into classes by molecular weight: HSP100, 90, 70, 60 and 27. Each class displays relatively specialized functions, although they often act work in concert to achieve an optimal outcome. Recently HSPs have been shown to have a role in cancer cell growth and survival and also as potential biomarkers and therapeutic targets (7–28). Our objective was therefore to determine whether MTC is
E. Soudry et al. / Annals of Diagnostic Pathology 26 (2017) 52–56
53
associated with elevated expression levels of HSP90, HSP70 and GRP78 compared to normal thyroid. These findings may shed a light on the role of these HSPs on the MTC's tumor biology and may have clinical implications for the prognostic analysis and treatment of patients with MTC.
2. Materials and methods Following approval of the study by the institutional review board (#4101), primary medullary thyroid carcinoma tissue specimens were obtained from 20 patients treated in our center (study group). Primary non cancerous thyroid tissues were obtained as a control group from 10 subjects with either follicular adenoma , colloid goiter or postmortem normal thyroid. Tissue specimens were verified to by a senior pathologist (S.M) to include MTC tissue for the study group or normal thyroid for the control group prior to proceeding with the immunohistochemical study.
2.1. Immunohistochemical study Tissue specimens were fixed with 10% formalin, embedded in paraffin, cut into 5-μm sections and placed on slides. The sections were deparaffinized, and endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol 100% for 25 min and washed with triethanolamine-buffered saline (TBS) at pH 7.6. For antigen activation, the sections were immersed in citrate buffer (pH 6), boiled, and incubated in a microwave oven at 95 °C for 10 min. After the sections were rinsed with TBS at pH 7.6, they were incubated in 20% swine serum (Wako, Osaka, Japan) for 20 min to block nonspecific reactions. The sections were then incubated overnight with the primary antibodies, HSP90, HSP70 and GRP78, at a dilution of 1:50, 1:40 and 1:500, respectively. The following morning, the sections were rinsed with TBS and incubated with peroxidase-labeled goat anti-mouse/rabbit antibody (Envision™) for 30 min. They were then rinsed again with TBS and incubated with 0.02% 3,3-diaminobezidinetetrahydrochloride (DAB) in 0.05 M Tris buffer for visualization of the peroxide reaction. This was followed by rinsing with tap water for 20 min and counterstaining with Mayer's hematoxylin. Finally, the sections were dehydrated by incubation with alcohol in rinsing dilutions (70%, 95% and 100% respectively) and cleared by incubation with toluene. Sections for negative controls were incubated with peroxidase-labeled goat antimouse\rabbit antibody, but without the primary antibody. Sections for positive controls were prepared from breast carcinoma for testing for HSP70 and GRP78 and from endometrial carcinoma for testing for HSP90; the tissues used are known to stain positively for the respective antibodies.
2.2. Immunohistochemical evaluation Immunohistochemistry evaluation was performed by an experienced head and neck pathologist (SM) . Immunostaining was verified with positive control specimens. Cells were considered positive when immunoreactivity was clearly observed in the cytoplasm. At least 1000 cells in 5 randomly selected fields were carefully monitored and classified into 4 categories: strong positivity 3 (+++), N66% positive cells; diffuse 2 (++), 33% to 66% positive cells; heterogeneous 1 (+), 10% to 33% positive cells; and negative 0 (−), b10% positive cells. Intensity was defined according to the color of staining: deep brown 3 (+++); brown 2 (++); yellowish brown 1 (+); or faint color 0. When the intensity of staining was not uniform across the specimen, an intermediate score was assigned (e.g., 0.5, 1.5, 2.5). Specimens from the 2 groups were compared for percentage of the area that stained positive and for the intensity of the positive staining. Only the tumor tissue was analyzed in the study group specimens.
Fig. 1. Histogram of heat shock protein immunohistochemical evaluation.
2.3. Statistical analysis Statistical analyses were performed using the nonparametric MannWhitney test. A p value of b 0.05 was considered statistically significant.
3. Results 3.1. Patient characteristics The study group consisted of 11 women and 9 men of average age 49 years at surgery (range: 9–80 years). Average tumor size was 2.3 cm (range: 0.8–5 cm). The control group consisted of 5 males and 5 females of average age 59 years.
3.2. Immunohistochemistry (Table 1, Fig. 2) Perinuclear staining was noted in all specimens and membrane staining was not found in any of the specimens (Fig. 1).
3.2.1. HSP90 On immunohistochemistry studies, the average score for percentage positively stained area for HSP90 was 1.65 in the study group and 1.3 in the control group. MTC specimens showed a 27% higher positivity rate than control specimens. The average staining intensity score was 0.98 in the study group and 0.95 in the control group. Neither of these differences was statistically significant (Table 1).
Table 1 Summary of immuno-histocehmical evaluation. Marker
Diagnosis
No.
Mean ± SD
P value
P value
HSP 70 area of staining
Medullary Normal Medullary Normal Medullary Normal Medullary Normal Medullary Normal Medullary Normal
20 10 20 10 20 10 20 10 20 10 20 10
2 ± 1.03 1.4 ± 0.7 1.08 ± 0.61 0.9 ± 0.39 1.65 ± 1.23 1.3 ± 0.67 0.98 ± 0.68 0.95 ± 0.37 3±0 2.8 ± 0.42 2.35 ± 0.46 2.35 ± 0.53
N.S
0.08
N.S
0.52
N.S
0.4
N.S
0.92
0.003
0.042
N.S
0.63
HSP 70 intensity HSP 90 area of staining HSP 90 intensity GRP 78 area of staining GRP 78 intensity
54
E. Soudry et al. / Annals of Diagnostic Pathology 26 (2017) 52–56
Fig. 2. HSP90, HSP70 and GRP78 immunostaining comparison between medullary thyroid carcinoma and normal thyroid tissue. Asterisks marking C-cell hyperplasia staining.
3.2.2. HSP70 For HSP70, the score for percentage of positively stained area was 2 in the study group and 1.4 in the control group. MTC specimens demonstrated a 43% higher positivity level than control specimens. The average intensity score was 1.08 in the study group and 0.9 in the control group. MTC specimens revealed a 20% higher intensity level than control specimens. These differences were not statistically significant, but there was a tendency for a higher positivity level of immunostaining in the MTC specimens (P = 0.08) (Table 1).
3.2.3. GRP78 Analysis of the percentage area stained positively for GRP78 yielded a score of 3 in the study group and 2.8 in the control group. MTC
specimens demonstrated a 7% higher positivity level, nonetheless the between-groups difference was statistically significant (p = 0.003). The average staining intensity score was 2.35 in both groups (p = NS) (Table 1). As opposed to other types of cancer cells, we did not find evidence of GRP78 immunostaining of tumor cell membranes.
4. Discussion HSPs may serve as attractive therapeutic targets and biologic markers in different cancers due to their central role in regulating oncogenes, apoptosis and chemotherapeutic resistance. HSP90 interacts with a large cadre of client proteins, many of which are critical signaling intermediates that regulate crucial aspects of cell biology, such as gene
E. Soudry et al. / Annals of Diagnostic Pathology 26 (2017) 52–56
transcription, cell cycle regulation, and the interplay between cells and their environment. These processes are essential for cell growth and survival and become deregulated in cancer [7]. Accordingly, elevated levels of HSP90 have been found in breast, colon, and lung cancer [8-11]. HSP90 is an attractive therapeutic target, on the assumption that its inhibition will inhibit essential processes for oncoprotein function and cancer cell survival. One HSP90 inhibitor, 17 allylamino-17demethoxyglendamycin (17AAG) is currently under investigation as a therapeutic agent for advanced staged thyroid cancers [12]. HSP70 acts under conditions of stress [9,13] in order to prevent cell apoptosis [9,14]. HSP70 is expressed by different cancer cells [9,13]. In some cancers, its expression has been associated with worse prognosis and chemoradiotherapy resistance [13,15-20]; in others, it has been associated with better prognosis [17,21]. Glucose-related protein 78 (GRP78) is sub-member of the HSP70 family, located mostly in the endoplasmic reticulum. Its expression rises in conditions of glucose deprivation. Recent studies have reported the presence of GRP78 on cancer cell membranes [22,23]. Cancers with elevated levels of GRP78 were found to be characterized by poorer differentiation, higher recurrence rates, worse prognosis, and resistance to chemotherapeutic agents [22,24]. Therefore, this protein, too, may serve as a biomarker and therapeutic target in cancer. Several groups have found that antiGRP78 antibodies inhibit the proliferation of several carcinoma cell lines [22,25,26], and that cytotoxic and antiangiogenic therapeutic agents can be inserted into tumor cells via membrane GRP78 [22,27,28]. GRP78 was found to regulate thyroid cancer cells sensitivity to antitumoral agents such as proteasome inhibitors [29]. HSP90 is the only family member that has been evaluated so far in thyroid cancers [30-32]. These studies demonstrated low levels of HSP90 in papillary thyroid carcinoma and a high sensitivity of anaplastic carcinoma cell lines to the HSP90 inhibitor 17AAG. 17AAG was found to induce cell cycle arrest in cell lines of MTC carrying RET oncogenic mutation [33]. However, a phase II, clinical trial of the effect of 17AAG therapy in patients with inoperable locoregionally advanced or metastatic differentiated or medullary thyroid cancer was discontinued due to disease progression and unacceptable toxicity [34]. Little is known about the association of HSP70 and GRP78 with thyroid tumors. In this study we analyzed the expression of HSPs in MTC using immunostaining of primary tumor and non cancerous thyroid tissues. We found intermediate intensity level of HSP90 immunostaining in MTC specimens. The level was 27% higher than in normal thyroid specimens, however this difference was not statistically significant. Further analysis of HSP70 immunostaining yielded a 43% higher level of positivity in the MTC specimens than in the controls, which nearly reached statistical significance, and a 20% higher intensity level which did not reach statistical significance. These results may imply that HSP70 and HSP90 may not have a significant role in MTC's tumor biology, nonetheless, since our sample size was limited, it is possible that in larger cohorts significant results may be reached. Analysis of GRP78 immunostaining revealed nonetheless that a significantly higher number of cells stained positively for GRP78 in the MTC specimens versus the controls. There was, however, no between-group difference in intensity (high in both). Several studies have shown that cancers with high levels of HSP70 and GRP78 are associated with chemotherapeutic resistance and disease aggressiveness. Our findings of significantly elevated GRP78 levels and higher HSP70 levels might imply a role for these proteins in the resistance of MTC to chemotherapy. Confirmation in larger studies is necessary and may give rise to new potential treatment strategies for MTC.
5. Conclusions In conclusion, the present preliminary study demonstrates higher expression levels, though not significant, of HSP90 and HSP70 and a significantly increased expression of GRP78 in MTC compared to controls. These findings suggest that these HSPs may have a role in MTC's tumor
55
biology and chemotherapeutic resistance and may also serve as potential therapeutic targets. Nevertheless, confirmation in larger cohorts is necessary to fully elucidate the association between HSPs and MTC. Conflict of interest The authors declare they have no conflict of interest. References [1] Ball DW. Medullary thyroid cancer: monitoring and therapy. Endocrinol Metab Clin N Am 2007;36:823–37. [2] de Groot JW, Links TP, Plukker JT, Lips CJ, Hofstra RM. RET as a diagnostic and therapeutic target in sporadic and hereditary endocrine tumors. Endocr Rev 2006;27: 535–60. [3] Saad M, Ordonez NG, Rashid RK, Guido JJ, Hill Jr CS, Hickey RC, et al. Medullary thyroid carcinoma: a study of the clinical features and prognostic factors in 161 patients. Medicine 1984;63:319–42. [4] Kakudo K, Carney JR, Sizemore GW. Medullary carcinoma of the thyroid: biological behavior of the sporadic and familial neoplasm. Cancer 1985;55:2818–21. [5] Nollen EA, Morimoto RI. Chaperoning signaling pathways: molecular chaperones as stress-sensing “heat shock” proteins. J Cell Sci 2002;115:2809–16. [6] Cullinan SB, Whitesell L. Heat shock protein 90: a unique chemotherapeutic target. Semin Oncol 2006;33:457–65. [7] Itoh H, Ogura M, Komatsuda A. A novel chaperone-activity-reducing mechanism of the 90-kDa molecular chaperone HSP90. Biochem J 1999;343:697–703. [8] Pratt WB, Toft DO. Regulation of signaling protein function and trafficking by the hsp90/hsp70-based chaperone machinery. Exp Biol Med (Maywood) 2003;228: 111–33. [9] Garrido C, et al. HSP 27 an HSP 70 – anti apoptotic proteins with tumorigenic properties. Cell Cycle 2006;5:2592–601. [10] Nanbu K, Konishi I, Mandai M. Prognostic significance of heat shock proteins HSP70 and HSP90 in endometrial carcinomas. Cancer Detect Prev 1998;22:549–55. [11] Kamal A, Thao L, Sensintaffar J. A high-affinity conformation of Hsp90 confers tumour selectivity on Hsp90 inhibitors. Nature 2003;425:407–10. [12] Kundra P, Burman KD. Thyroid cancer molecular signaling pathways and use of targeted therapy. Endocrinol Metab Clin N Am 2007;36:839–53. [13] Beckmann RP, Mizzen LE, Welch WJ. Interaction of Hsp 70 with newly synthesized proteins: implications for protein folding and assembly. Science 1990;248:850–4. [14] Li CY, Lee JS, Ko YG, Kim JI, Seo JS. Heat shock protein 70 inhibits apoptosis downstream of cytochrome c release and upstream of caspase-3 activation. J Biol Chem 2000;275:25665–71. [15] Gurbuxani S, Schmitt E, Cande C, Parcellier A, Hammann A, Daugas E, et al. Heat shock protein 70 binding inhibits the nuclear import of apoptosis-inducing factor. Oncogene 2003;22:6669–78. [16] Santarosa M, Favaro D, Quaia M, Galligioni E. Expression of heat shock protein 72 in renal cell carcinoma: possible role and prognostic implications in cancer patients. Eur J Cancer 1997;33:873–7. [17] Nanbu K, Konishi I, Mandai M, Kuroda H, Hamid AA, Komatsu T, et al. Prognostic significance of heat shock proteins HSP70 and HSP90 in endometrial carcinomas. Cancer Detect Prev 1998;22:549–55. [18] Ciocca DR, Oesterreich S, Chamness GC, McGuire WL, Fuqua SA. Biological and clinical implications of heat shock protein 27,000 (Hsp27): a review. J Natl Cancer Inst 1993;85:1558–70. [19] Vargas-Roig LM, Gago FE, Tello O, Aznar JC, Ciocca DR. Heat shock protein expression and drug resistance in breast cancer patients treated with induction chemotherapy. Int J Cancer 1998;79:468–75. [20] Brondani Da Rocha A, Regner A, Grivicich I, Pretto Schunemann D, Diel C, Kovaleski G, et al. Radioresistance is associated to increased Hsp70 content in human glioblastoma cell lines. Int J Oncol 2004;25:777–85. [21] Trieb K, Lechleitner T, Lang S, Windhager R, Kotz R, Dirnhofer S. Heat shock protein 72 expression in osteosarcomas correlates with good response to neoadjuvant chemotherapy. Hum Pathol 1998;29:1050–5. [22] Lee AS. GRP78 induction in cancer: therapeutic and prognostic implications. Cancer Res 2007;67:3496–9. [23] Shin BL, Wang H, Yim AM, Le Naour F, Brichory F, Jang JH, et al. Global profiling of the cell surface proteome of cancer cells uncovers the abundance of proteins with chaperone function. J Biol Chem 2003;278:7607–16. [24] Kim Y, Lillo AM, Steiniger SC, Liu Y, Ballatore C, Anichini A, et al. Targeting heat shock protein on cancer cells: selection, characterization, and cell penetrating properties of peptidic GRP78 ligand. Biochemistry 2006;45:9434–44. [25] Gronow MG, Kaczowka SJ, Payne S, Wang F, Gawdi G, Pizzo SV. Plasminogen-structural domains exhibit different functions when associated with cell surface GRP78 or the voltage-dependent anion channel. J Biol Chem 2007;282:32811–20. [26] Gronow MG, Cuchacovich M, Llanos C, Urzua C, Gawdi G, Pizzo SV. Prostate cancer cell proliferation in vitro is modulated by antibodies against GRP 78 isolated from patient serum. Cancer Res 2006;66:11424–31. [27] Davidson DJ, Haskell C, Majest S, Kherzai A, Egan DA, Walter KA, et al. Kringle 5 of human plasminogen induces apoptosis of endothelial and tumor cells through surface-expressed glucose-regulated protein 78. Cancer Res 2005;65:4663–72. [28] Kim Y, Lillo AM, Steiniger SC, Liu Y, Ballatore C, Anichini A, et al. Targeting heat shock proteins on cancer cells: selection, characterization, and cell-penetrating properties of a peptidic GRP78 ligand. Biochemistry 2006;45:9434–44.
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[29] Wang H-Q, Du Z-X, Zhang H-Y, Gao D-X. Different induction of GRP78 and CHOP as a predictor of sensitivity to proteasome inhibitors in thyroid cancer cells. Endocrinology 2011(7):148. http://dx.doi.org/10.1210/en.2006-1564. [30] Wallin G, et al. Expression of the thyroid hormone receptor, the oncogenes c-myc and H-ras, and the 90 kD heat shock protein in normal, hyperplasitc, and neoplastic human thyroid tissue. Thyroid 1992;2:307–13. [31] Braga-Basaria M, Hardy E, Gottfried R, Burman KD, Saji M, Ringel MD. 17Allylamino-17-demethoxygeldanamycin activity against thyroid cancer cell lines correlates with heat shock protein 90 levels. J Clin Endocrinol Metab 2004;89(6): 2982–8.
[32] Park JW, Yeh MW, Wong MG, Lobo M, Hyun WC, Duh QY, et al. The heat shock protein 90-binding geldanamycin inhibits cancer cell proliferation, down-regulates oncoproteins, and inhibits epidermal growth factor-induced invasion in thyroid cancer cell lines. J Clin Endocrinol Metab 2003;88(7):3346–53. [33] Alfano L, Guida T, Provitera L, Vecchio G, Billaud M, Santoro M, et al. RET is a heat shock protein 90 (HSP90) client protein and is knocked down upon HSP90 pharmacological block. J Clin Endocrinol Metab 2011 [published online April 30]. [34] Moley JF, Adkins D, Bible KC, Traynor AM, Molina JR, Colon-Otero G, et al. 17Allylaminogeldanamycin in advanced medullary and differentiated thyroid carcinoma. J Clin Oncol 2011;29(15_suppl):5582.
Contents lists available at ScienceDirect
Annals of Diagnostic Pathology
Heat shock proteins HSP90, HSP70 and GRP78 expression in medullary thyroid carcinoma Ethan Soudry a,d,⁎, Sagit Stern Shavit a,d, Britta Hardy c,d, Sarah Morgenstern b,d, Tuvia Hadar a,d, Raphael Feinmesser a,d a
Department of Otolaryngology - Head and Neck Surgery, Beilinson Campus, Petah Tiqwa, Israel Department of Pathology, Rabin Medical Center, Beilinson Campus, Petah Tiqwa, Israel c Felsenstein Research Medical Center, Beilinson Campus, Petah Tiqwa, Israel d Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel b
a r t i c l e
i n f o
Keywords: Medullary thyroid carcinoma Heat shock protein 70 Heat shock protein 90 GRP 78 Immunohistochemistry
a b s t r a c t Background: Medullary thyroid carcinoma management consists mainly of surgical resection and is largely chemoresistant. There is ongoing effort to discover novel therapies for medullary thyroid carcinoma. Increased levels of heat shock proteins have been associated with multiple cancers and are being studied as potential therapeutic targets. The purpose of this study was to determine the expression levels of heat shock proteins 90 and 70 and of glucose related protein 78 in medullary thyroid carcinoma tissues compared with normal thyroid tissues. Methods: 20 tissue specimens of medullary thyroid carcinoma and 10 specimens of thyroids without malignancy were analyzed by immunohistochemistry. Results: Medullary thyroid carcinoma specimens showed 27% higher expression level of heat shock protein 90 immunostaining, and a 43% higher expression level of heat shock protein 70 immunostaining versus normal controls. These differences, however, were not statistically significant. A significantly higher expression level was noted for glucose related protein 78 in the medullary thyroid carcinoma specimens than in the controls. Conclusion: This study indicates increased expression levels of heat shock proteins 90 and 70 and glucose related protein 78 levels in medullary thyroid carcinoma. These findings, though preliminary imply that these proteins may have a role in medullary thyroid carcinoma's tumor biology and may have and future therapeutic options. Larger cohorts are needed to corroborate these results. © 2016 Elsevier Inc. All rights reserved.
1. Introduction Medullary thyroid carcinoma (MTC) is an endocrine neoplasm that arises in the parafollicular C cells of the thyroid gland and may secrete calcitonin and carcinoembryonic antigen. It accounts for 2% to 5% of all thyroid carcinomas [1]. Seventy-five percent of cases are sporadic and the rest are inherited. Up to 50% of sporadic cases and 95% of inherited cases are associated with mutations in the RET proto-oncogene [2]. The clinical course is less aggressive than in anaplastic carcinomas but more aggressive than in well-differentiated thyroid cancers. Regional and distant metastases are frequent (up to 50% of cases). Current management is mainly consisted of surgical resection which includes total thyroidectomy and lymph-node dissection. Radioactive iodine treatment is not effective because, owing to their parafolliccular C-cell origin, MTCs do not take up iodine. External beam radiation therapy has also not been found effective, and there is no effective chemotherapy regimen [1,3]. ⁎ Corresponding author at: Department of Otorhinolaryngology - Head and Neck Surgery, Rabin Medical Center - Beilinson Hospital, Petach Tikva 49100, Israel. E-mail address: [email protected] (E. Soudry).
http://dx.doi.org/10.1016/j.anndiagpath.2016.11.003 1092-9134/© 2016 Elsevier Inc. All rights reserved.
The prognosis of patients with MTC is directly related to disease stage. The overall 10-year survival rate is 61% to 75%, but it decreases to 45% if cervical nodes are involved [3,4]. Therefore, researchers are seeking promising new therapeutic modalities for patients with MTC unresponsive to traditional therapy. Potential targets are thyroid molecular signaling pathways and factors associated with cancer cell biology including vascular endothelial growth factor receptors and antibodies, angiogenesis inhibitors, tyrosine kinase inhibitors, and heat shock protein (HSP) inhibitors [1]. In 1963, HSPs were identified as a unique family of proteins that are expressed following exposure to protein-damaging environmental stresses to promote protein renaturation, restore homeostasis and enhance survival. The process is termed the heat shock response [5,6]. The heat shock family of proteins is divided into classes by molecular weight: HSP100, 90, 70, 60 and 27. Each class displays relatively specialized functions, although they often act work in concert to achieve an optimal outcome. Recently HSPs have been shown to have a role in cancer cell growth and survival and also as potential biomarkers and therapeutic targets (7–28). Our objective was therefore to determine whether MTC is
E. Soudry et al. / Annals of Diagnostic Pathology 26 (2017) 52–56
53
associated with elevated expression levels of HSP90, HSP70 and GRP78 compared to normal thyroid. These findings may shed a light on the role of these HSPs on the MTC's tumor biology and may have clinical implications for the prognostic analysis and treatment of patients with MTC.
2. Materials and methods Following approval of the study by the institutional review board (#4101), primary medullary thyroid carcinoma tissue specimens were obtained from 20 patients treated in our center (study group). Primary non cancerous thyroid tissues were obtained as a control group from 10 subjects with either follicular adenoma , colloid goiter or postmortem normal thyroid. Tissue specimens were verified to by a senior pathologist (S.M) to include MTC tissue for the study group or normal thyroid for the control group prior to proceeding with the immunohistochemical study.
2.1. Immunohistochemical study Tissue specimens were fixed with 10% formalin, embedded in paraffin, cut into 5-μm sections and placed on slides. The sections were deparaffinized, and endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol 100% for 25 min and washed with triethanolamine-buffered saline (TBS) at pH 7.6. For antigen activation, the sections were immersed in citrate buffer (pH 6), boiled, and incubated in a microwave oven at 95 °C for 10 min. After the sections were rinsed with TBS at pH 7.6, they were incubated in 20% swine serum (Wako, Osaka, Japan) for 20 min to block nonspecific reactions. The sections were then incubated overnight with the primary antibodies, HSP90, HSP70 and GRP78, at a dilution of 1:50, 1:40 and 1:500, respectively. The following morning, the sections were rinsed with TBS and incubated with peroxidase-labeled goat anti-mouse/rabbit antibody (Envision™) for 30 min. They were then rinsed again with TBS and incubated with 0.02% 3,3-diaminobezidinetetrahydrochloride (DAB) in 0.05 M Tris buffer for visualization of the peroxide reaction. This was followed by rinsing with tap water for 20 min and counterstaining with Mayer's hematoxylin. Finally, the sections were dehydrated by incubation with alcohol in rinsing dilutions (70%, 95% and 100% respectively) and cleared by incubation with toluene. Sections for negative controls were incubated with peroxidase-labeled goat antimouse\rabbit antibody, but without the primary antibody. Sections for positive controls were prepared from breast carcinoma for testing for HSP70 and GRP78 and from endometrial carcinoma for testing for HSP90; the tissues used are known to stain positively for the respective antibodies.
2.2. Immunohistochemical evaluation Immunohistochemistry evaluation was performed by an experienced head and neck pathologist (SM) . Immunostaining was verified with positive control specimens. Cells were considered positive when immunoreactivity was clearly observed in the cytoplasm. At least 1000 cells in 5 randomly selected fields were carefully monitored and classified into 4 categories: strong positivity 3 (+++), N66% positive cells; diffuse 2 (++), 33% to 66% positive cells; heterogeneous 1 (+), 10% to 33% positive cells; and negative 0 (−), b10% positive cells. Intensity was defined according to the color of staining: deep brown 3 (+++); brown 2 (++); yellowish brown 1 (+); or faint color 0. When the intensity of staining was not uniform across the specimen, an intermediate score was assigned (e.g., 0.5, 1.5, 2.5). Specimens from the 2 groups were compared for percentage of the area that stained positive and for the intensity of the positive staining. Only the tumor tissue was analyzed in the study group specimens.
Fig. 1. Histogram of heat shock protein immunohistochemical evaluation.
2.3. Statistical analysis Statistical analyses were performed using the nonparametric MannWhitney test. A p value of b 0.05 was considered statistically significant.
3. Results 3.1. Patient characteristics The study group consisted of 11 women and 9 men of average age 49 years at surgery (range: 9–80 years). Average tumor size was 2.3 cm (range: 0.8–5 cm). The control group consisted of 5 males and 5 females of average age 59 years.
3.2. Immunohistochemistry (Table 1, Fig. 2) Perinuclear staining was noted in all specimens and membrane staining was not found in any of the specimens (Fig. 1).
3.2.1. HSP90 On immunohistochemistry studies, the average score for percentage positively stained area for HSP90 was 1.65 in the study group and 1.3 in the control group. MTC specimens showed a 27% higher positivity rate than control specimens. The average staining intensity score was 0.98 in the study group and 0.95 in the control group. Neither of these differences was statistically significant (Table 1).
Table 1 Summary of immuno-histocehmical evaluation. Marker
Diagnosis
No.
Mean ± SD
P value
P value
HSP 70 area of staining
Medullary Normal Medullary Normal Medullary Normal Medullary Normal Medullary Normal Medullary Normal
20 10 20 10 20 10 20 10 20 10 20 10
2 ± 1.03 1.4 ± 0.7 1.08 ± 0.61 0.9 ± 0.39 1.65 ± 1.23 1.3 ± 0.67 0.98 ± 0.68 0.95 ± 0.37 3±0 2.8 ± 0.42 2.35 ± 0.46 2.35 ± 0.53
N.S
0.08
N.S
0.52
N.S
0.4
N.S
0.92
0.003
0.042
N.S
0.63
HSP 70 intensity HSP 90 area of staining HSP 90 intensity GRP 78 area of staining GRP 78 intensity
54
E. Soudry et al. / Annals of Diagnostic Pathology 26 (2017) 52–56
Fig. 2. HSP90, HSP70 and GRP78 immunostaining comparison between medullary thyroid carcinoma and normal thyroid tissue. Asterisks marking C-cell hyperplasia staining.
3.2.2. HSP70 For HSP70, the score for percentage of positively stained area was 2 in the study group and 1.4 in the control group. MTC specimens demonstrated a 43% higher positivity level than control specimens. The average intensity score was 1.08 in the study group and 0.9 in the control group. MTC specimens revealed a 20% higher intensity level than control specimens. These differences were not statistically significant, but there was a tendency for a higher positivity level of immunostaining in the MTC specimens (P = 0.08) (Table 1).
3.2.3. GRP78 Analysis of the percentage area stained positively for GRP78 yielded a score of 3 in the study group and 2.8 in the control group. MTC
specimens demonstrated a 7% higher positivity level, nonetheless the between-groups difference was statistically significant (p = 0.003). The average staining intensity score was 2.35 in both groups (p = NS) (Table 1). As opposed to other types of cancer cells, we did not find evidence of GRP78 immunostaining of tumor cell membranes.
4. Discussion HSPs may serve as attractive therapeutic targets and biologic markers in different cancers due to their central role in regulating oncogenes, apoptosis and chemotherapeutic resistance. HSP90 interacts with a large cadre of client proteins, many of which are critical signaling intermediates that regulate crucial aspects of cell biology, such as gene
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transcription, cell cycle regulation, and the interplay between cells and their environment. These processes are essential for cell growth and survival and become deregulated in cancer [7]. Accordingly, elevated levels of HSP90 have been found in breast, colon, and lung cancer [8-11]. HSP90 is an attractive therapeutic target, on the assumption that its inhibition will inhibit essential processes for oncoprotein function and cancer cell survival. One HSP90 inhibitor, 17 allylamino-17demethoxyglendamycin (17AAG) is currently under investigation as a therapeutic agent for advanced staged thyroid cancers [12]. HSP70 acts under conditions of stress [9,13] in order to prevent cell apoptosis [9,14]. HSP70 is expressed by different cancer cells [9,13]. In some cancers, its expression has been associated with worse prognosis and chemoradiotherapy resistance [13,15-20]; in others, it has been associated with better prognosis [17,21]. Glucose-related protein 78 (GRP78) is sub-member of the HSP70 family, located mostly in the endoplasmic reticulum. Its expression rises in conditions of glucose deprivation. Recent studies have reported the presence of GRP78 on cancer cell membranes [22,23]. Cancers with elevated levels of GRP78 were found to be characterized by poorer differentiation, higher recurrence rates, worse prognosis, and resistance to chemotherapeutic agents [22,24]. Therefore, this protein, too, may serve as a biomarker and therapeutic target in cancer. Several groups have found that antiGRP78 antibodies inhibit the proliferation of several carcinoma cell lines [22,25,26], and that cytotoxic and antiangiogenic therapeutic agents can be inserted into tumor cells via membrane GRP78 [22,27,28]. GRP78 was found to regulate thyroid cancer cells sensitivity to antitumoral agents such as proteasome inhibitors [29]. HSP90 is the only family member that has been evaluated so far in thyroid cancers [30-32]. These studies demonstrated low levels of HSP90 in papillary thyroid carcinoma and a high sensitivity of anaplastic carcinoma cell lines to the HSP90 inhibitor 17AAG. 17AAG was found to induce cell cycle arrest in cell lines of MTC carrying RET oncogenic mutation [33]. However, a phase II, clinical trial of the effect of 17AAG therapy in patients with inoperable locoregionally advanced or metastatic differentiated or medullary thyroid cancer was discontinued due to disease progression and unacceptable toxicity [34]. Little is known about the association of HSP70 and GRP78 with thyroid tumors. In this study we analyzed the expression of HSPs in MTC using immunostaining of primary tumor and non cancerous thyroid tissues. We found intermediate intensity level of HSP90 immunostaining in MTC specimens. The level was 27% higher than in normal thyroid specimens, however this difference was not statistically significant. Further analysis of HSP70 immunostaining yielded a 43% higher level of positivity in the MTC specimens than in the controls, which nearly reached statistical significance, and a 20% higher intensity level which did not reach statistical significance. These results may imply that HSP70 and HSP90 may not have a significant role in MTC's tumor biology, nonetheless, since our sample size was limited, it is possible that in larger cohorts significant results may be reached. Analysis of GRP78 immunostaining revealed nonetheless that a significantly higher number of cells stained positively for GRP78 in the MTC specimens versus the controls. There was, however, no between-group difference in intensity (high in both). Several studies have shown that cancers with high levels of HSP70 and GRP78 are associated with chemotherapeutic resistance and disease aggressiveness. Our findings of significantly elevated GRP78 levels and higher HSP70 levels might imply a role for these proteins in the resistance of MTC to chemotherapy. Confirmation in larger studies is necessary and may give rise to new potential treatment strategies for MTC.
5. Conclusions In conclusion, the present preliminary study demonstrates higher expression levels, though not significant, of HSP90 and HSP70 and a significantly increased expression of GRP78 in MTC compared to controls. These findings suggest that these HSPs may have a role in MTC's tumor
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