Clin Oral Invest DOI 10.1007/s00784-014-1341-z
ORIGINAL ARTICLE
Prognostic significance of neural stem cell markers, Nestin and Musashi-1, in oral squamous cell carcinoma: expression pattern of Nestin in the precancerous stages of oral squamous epithelium Gokulan Ravindran & Halagowder Devaraj
Received: 15 April 2014 / Accepted: 19 October 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Background Besides the tissue-specific stem cell markers, neural and hematopoietic stem cell markers were found to play an important role in carcinogenesis. Based on this background, we have investigated the expression pattern and prognostic significance of neural stem cell markers, Nestin and Musashi-1, in oral cancer. Methods We used immunohistochemistry and immunofluorescence analyses to study the expression pattern and correlation between Nestin and Musashi-1 in oral squamous cell carcinoma. The Kaplan-Meier method was used to construct overall and disease-free survival curves, and the differences were calculated using log-rank test. Results Nestin expression was gradually increased in the transformation stages of oral cancer. Both Nestin and Musashi-1 expressions were associated with higher stage and poorly differentiated status of oral carcinoma. Interestingly, Nestin and Musashi-1 double positive cases showed statistically highly significant correlation with poorer survival of oral carcinoma patients. Conclusions Expression of Nestin in the preneoplastic lesions indicates its role in the transformation of oral squamous Electronic supplementary material The online version of this article (doi:10.1007/s00784-014-1341-z) contains supplementary material, which is available to authorized users. G. Ravindran : H. Devaraj (*) Unit of Biochemistry, Department of Zoology, University of Madras, Guindy Campus, Chennai, Tamil Nadu, India e-mail: [email protected] H. Devaraj e-mail: [email protected] Present Address: G. Ravindran Department of Oral Biology and Biomedical Sciences, Faculty of Dentistry, University of Malaya, Kuala Lumpur, Malaysia
epithelium. Clinicopathological and survival analyses suggest that Nestin and Musashi-1 might be associated with invasion, differentiation and poorer survival in oral squamous cell carcinoma. In addition to their role as independent prognostic indicators, Nestin and Musashi-1 double positivity can be used to select high-risk cases for effective therapy and this is the novel finding of this study. Clinical relevance Nestin and Musashi-1 are found to be independent prognostic markers of oral cancer, and they might be used as molecular targets for effective therapy. Keywords Oral squamous cell carcinoma . Nestin . Cancer stem cells . Musashi-1 . Neural stem cell markers
Introduction Oral cancer ranks sixth among the most prevalent cancers worldwide [1]. Cancer is a combination of several functional abnormalities including activation of oncogenes, loss of tumour suppressor genes, increased angiogenesis, decreased apoptosis, acquired replicative potential, invasion and metastasis [2]. Recently, it was found that only a subpopulation of cells within the tumour tissue has the capacity to drive tumour progression. These cells seed new tumours in immunodeficient mice in vivo. In addition, these cells share markers that are also expressed in adult tissue-specific stem cells. Therefore, they are termed as cancer stem cells [3, 4]. Furthermore, epithelial to mesenchymal transition (EMT) plays an important role in metastasis. The cells obtained through EMT were found to express stem cell-related markers and carry mutations which induce tumours in other organs [5]. Cancer stem cells are believed to be involved in the recurrence of tumours due to their resistance to chemotherapy [3]. Reexpression of stem cell markers in carcinoma provides a
Clin Oral Invest
platform for scientists to explore the possible involvement of stem cell molecules during the multistep process of carcinogenesis. In addition to the tissue-specific stem cell-related markers, neural and hematopoietic stem/progenitor cell markers have been found to be expressed in higher levels in tumours including oral squamous cell carcinoma [6, 7]. Nestin is a class VI intermediate filament protein predominantly expressed in the progenitor cells of differentiated tissues [8]. Initially, Nestin was identified as a stem/progenitor cell marker during the development of the central nervous system (CNS) and it gets downregulated upon differentiation [9, 10]. Apart from the CNS, Nestin expression was also found in progenitor cells of skeletal muscle, heart, testis, pancreas, developing tooth bud, skin, hair follicle and blood vessels [11–14]. Interestingly, it has been observed that Nestin gets re-expressed after injury and in regenerating tissues [11]. Nestin is expressed in several tumours [15, 16], and its prognostic significance has been demonstrated in breast cancer, lung cancer, melanoma, glioma and pancreatic cancer [17–21]. Nestin has been found to regulate invasion, metastasis, cell cycle and apoptosis in pancreatic ductal adenocarcinoma and lung adenocarcinoma through regulation of cytoskeletal proteins and stemness [15, 22]. Oral cancer stem-like cells also show a high level of Nestin expression [6]. Nestin has several interacting partners like N-Myc, Sox 1/2/3, Oct-4, Vimentin and Cdk5 [16]. Nestin has been found to regulate PI3K signaling in neural progenitor cells [23]. Moreover, downregulation of Nestin is found to suppress phosphorylation of AKT in melanoma cells [21, 24]. In neuroblastoma cells, Nestin was found to interact with the nuclear DNA and modulate the aggressive behavior of the tumour [25]. Recently, it was found that the CD24+/Nestin+ cancer stem cells induce sphere formation in cervical carcinoma cells [16, 26]. Musashi, which belongs to a group of highly conserved proteins, was identified in Drosophila. In mammals, there are two homologs of Musashi protein, viz. Musashi-1 and Musashi-2 [27–29]. Musashi-1 is a RNA-binding protein which regulates mRNA translation. Similar to Nestin, Musashi-1 expression was observed in stem/progenitor cells of the CNS. In addition to the CNS, Musashi-1 was also found in adult stem cells of the eye, intestine, stomach, mammary gland and hair follicle. Nevertheless, Musashi-1 has been found to be expressed in higher levels in many tumours such as glioma, colorectal adenoma, hepatoma, non-small lung cancer, retinoblastoma, melanoma and cervical cancer [30–35]. Musashi-1 maintains proliferation of gastric carcinoma cells as evidenced by its positive correlation with PCNA [36]. Musashi-1 controls the Notch signaling cascade by inhibiting the activity of its repressor m-Numb. Interestingly, p21WAF1 is also found to be a target of Musashi-1 [37, 38]. Musashi-1 plays a significant role in several neurodegenerative disorders including Alzheimer’s disease and epilepsy [31,
39]. Particularly, Musashi regulates insulin expression and proliferation in diabetes as a consequence of endoplasmic reticulum-related stress [40]. Recently, studies reveal a stimulatory role of Musashi-1 in inducing WNT pathway through the activation of Frat-1. Musashi-1 induces the proliferation of mammary gland cells and regulates the growth of colon carcinoma. Notably, Musashi-1 maintains progenitor cell homeostasis by regulating Wnt, Notch and thyroid pathways which are affected during colon tumourigenesis [41]. Musashi-1 is also found to be expressed in cancer stem cells. Additionally, the knockdown of Musashi-1 results in decreased proliferation, growth arrest and apoptosis in a number of tumours including colon adenocarcinoma [42, 43]. Moreover, Musashi-1 has been found to be highly expressed in preneoplastic and neoplastic tissues of oral squamous cell carcinoma [7]. In our previous study, we have investigated the expression pattern of Musashi-1 in preneoplastic and neoplastic oral squamous cell carcinoma tissues. However, the prognostic significance of Musashi-1 was uncovered in oral cancer. To our knowledge, the expression of Nestin has not been demonstrated in the multistep process of oral carcinogenesis. Moreover, its prognostic significance was not studied extensively in oral cancer. Based on this background, we have examined the expression pattern of Nestin in the progressive stages of oral squamous cell carcinoma. The association between Nestin and Musashi-1 expression was also analysed in oral carcinoma tissues. Furthermore, the prognostic significance of both Musashi-1 and Nestin has been investigated to explore the possibility of using both markers for worst prognosis and therapeutic purposes.
Materials and methods Tissue samples Tissue samples from precancerous lesions and carcinoma [mild–moderate dysplasia (36), severe dysplasia (28) and oral squamous cell carcinoma (72)] were obtained from the Royapettah Hospital, Chennai. In addition, we have collected 15 normal samples from patients undergoing orthodontic surgery. The samples have been fixed immediately with 10 % buffered formalin and they were processed for paraffin embedding. The sections were cut at 4 μm and mounted on coated glass slides. The histological grading of dysplasia and oral squamous cell carcinoma have been assessed according to the criteria illustrated by Pindborg et al. [44] by two pathologists until a consensus was reached. The range of follow-up is 12–50 months with a mean follow-up period of 31.7. The clearance was obtained from the Hospital Medical Board with permission from the Directorate of Medical Education, Government of Tamil Nadu, India (No. 21823/ME 1/1/2008).
Clin Oral Invest
Immunohistochemistry
Table 1 Expression of Nestin in precancerous and cancerous tissues of oral squamous epithelium
Immunohistochemistry was done on tissue sections as described previously [7]. The sections were deparaffinized in xylene and rehydrated through graded alcohols, and the antigen retrieval was done by heating the slides in 0.1 M sodium citrate buffer (pH 6.0) in a microwave oven. After cooling, the endogenous peroxidase activity was blocked by placing the slides in 3 % hydrogen peroxide for 10 min. The non-specific binding sites were blocked with 0.3 % bovine serum albumin (BSA) for 30 min at room temperature. The sections were then incubated (overnight at 4 °C) with mouse monoclonal antiMusashi-1 (Santa Cruz Biotechnology, USA) and goat polyclonal anti-Nestin (Santa Cruz Biotechnology, USA) in a dilution of 1:200 for the above-mentioned antibodies. The slides were immunostained using the respective horseradish peroxidase-conjugated secondary antibodies (Invitrogen, USA). The chromogen 3,3′-diaminobenzidine was then used as a substrate for localizing the antibody binding. After counterstaining with haematoxylin, the sections were mounted and viewed under a microscope. Negative controls without primary antibody were included for each staining. The immunostained sections were scored according to the percentage of stained cells in more than three random areas of cancer tissue as examined in ×200 magnification. Two independent observers without prior knowledge on the patient’s clinicopathological data assessed the immunostained slides. The staining was assessed as 0 (negative), no staining or staining in less than 5 % of cells; 1+ (mild), staining in 5–15 % of carcinoma cells; 2+ (moderate), staining in 16–25 % of carcinoma cells; and 3+ (intense), staining in >25 % of carcinoma
No. of patients
Fig. 1 Immunostaining of Nestin in the normal (a), mild dysplasia (b), severe dysplasia (c) and carcinoma (d) (inset: cytoplasmic staining of Musashi-1 in oral carcinoma cells) tissues of oral squamous epithelium. Bar= 100 μm
Nestin 0
1+
2+
3+
Normal
15
13
2
0
0
Mild–moderate Severe Carcinoma
36 28 72
11 4 7
14 7 13
9 8 22
2 9 30a
0 (negative), no staining or 25 % positive cells a
Expression of Nestin shows a significant difference between different groups calculated by chi-square (p
ORIGINAL ARTICLE
Prognostic significance of neural stem cell markers, Nestin and Musashi-1, in oral squamous cell carcinoma: expression pattern of Nestin in the precancerous stages of oral squamous epithelium Gokulan Ravindran & Halagowder Devaraj
Received: 15 April 2014 / Accepted: 19 October 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Background Besides the tissue-specific stem cell markers, neural and hematopoietic stem cell markers were found to play an important role in carcinogenesis. Based on this background, we have investigated the expression pattern and prognostic significance of neural stem cell markers, Nestin and Musashi-1, in oral cancer. Methods We used immunohistochemistry and immunofluorescence analyses to study the expression pattern and correlation between Nestin and Musashi-1 in oral squamous cell carcinoma. The Kaplan-Meier method was used to construct overall and disease-free survival curves, and the differences were calculated using log-rank test. Results Nestin expression was gradually increased in the transformation stages of oral cancer. Both Nestin and Musashi-1 expressions were associated with higher stage and poorly differentiated status of oral carcinoma. Interestingly, Nestin and Musashi-1 double positive cases showed statistically highly significant correlation with poorer survival of oral carcinoma patients. Conclusions Expression of Nestin in the preneoplastic lesions indicates its role in the transformation of oral squamous Electronic supplementary material The online version of this article (doi:10.1007/s00784-014-1341-z) contains supplementary material, which is available to authorized users. G. Ravindran : H. Devaraj (*) Unit of Biochemistry, Department of Zoology, University of Madras, Guindy Campus, Chennai, Tamil Nadu, India e-mail: [email protected] H. Devaraj e-mail: [email protected] Present Address: G. Ravindran Department of Oral Biology and Biomedical Sciences, Faculty of Dentistry, University of Malaya, Kuala Lumpur, Malaysia
epithelium. Clinicopathological and survival analyses suggest that Nestin and Musashi-1 might be associated with invasion, differentiation and poorer survival in oral squamous cell carcinoma. In addition to their role as independent prognostic indicators, Nestin and Musashi-1 double positivity can be used to select high-risk cases for effective therapy and this is the novel finding of this study. Clinical relevance Nestin and Musashi-1 are found to be independent prognostic markers of oral cancer, and they might be used as molecular targets for effective therapy. Keywords Oral squamous cell carcinoma . Nestin . Cancer stem cells . Musashi-1 . Neural stem cell markers
Introduction Oral cancer ranks sixth among the most prevalent cancers worldwide [1]. Cancer is a combination of several functional abnormalities including activation of oncogenes, loss of tumour suppressor genes, increased angiogenesis, decreased apoptosis, acquired replicative potential, invasion and metastasis [2]. Recently, it was found that only a subpopulation of cells within the tumour tissue has the capacity to drive tumour progression. These cells seed new tumours in immunodeficient mice in vivo. In addition, these cells share markers that are also expressed in adult tissue-specific stem cells. Therefore, they are termed as cancer stem cells [3, 4]. Furthermore, epithelial to mesenchymal transition (EMT) plays an important role in metastasis. The cells obtained through EMT were found to express stem cell-related markers and carry mutations which induce tumours in other organs [5]. Cancer stem cells are believed to be involved in the recurrence of tumours due to their resistance to chemotherapy [3]. Reexpression of stem cell markers in carcinoma provides a
Clin Oral Invest
platform for scientists to explore the possible involvement of stem cell molecules during the multistep process of carcinogenesis. In addition to the tissue-specific stem cell-related markers, neural and hematopoietic stem/progenitor cell markers have been found to be expressed in higher levels in tumours including oral squamous cell carcinoma [6, 7]. Nestin is a class VI intermediate filament protein predominantly expressed in the progenitor cells of differentiated tissues [8]. Initially, Nestin was identified as a stem/progenitor cell marker during the development of the central nervous system (CNS) and it gets downregulated upon differentiation [9, 10]. Apart from the CNS, Nestin expression was also found in progenitor cells of skeletal muscle, heart, testis, pancreas, developing tooth bud, skin, hair follicle and blood vessels [11–14]. Interestingly, it has been observed that Nestin gets re-expressed after injury and in regenerating tissues [11]. Nestin is expressed in several tumours [15, 16], and its prognostic significance has been demonstrated in breast cancer, lung cancer, melanoma, glioma and pancreatic cancer [17–21]. Nestin has been found to regulate invasion, metastasis, cell cycle and apoptosis in pancreatic ductal adenocarcinoma and lung adenocarcinoma through regulation of cytoskeletal proteins and stemness [15, 22]. Oral cancer stem-like cells also show a high level of Nestin expression [6]. Nestin has several interacting partners like N-Myc, Sox 1/2/3, Oct-4, Vimentin and Cdk5 [16]. Nestin has been found to regulate PI3K signaling in neural progenitor cells [23]. Moreover, downregulation of Nestin is found to suppress phosphorylation of AKT in melanoma cells [21, 24]. In neuroblastoma cells, Nestin was found to interact with the nuclear DNA and modulate the aggressive behavior of the tumour [25]. Recently, it was found that the CD24+/Nestin+ cancer stem cells induce sphere formation in cervical carcinoma cells [16, 26]. Musashi, which belongs to a group of highly conserved proteins, was identified in Drosophila. In mammals, there are two homologs of Musashi protein, viz. Musashi-1 and Musashi-2 [27–29]. Musashi-1 is a RNA-binding protein which regulates mRNA translation. Similar to Nestin, Musashi-1 expression was observed in stem/progenitor cells of the CNS. In addition to the CNS, Musashi-1 was also found in adult stem cells of the eye, intestine, stomach, mammary gland and hair follicle. Nevertheless, Musashi-1 has been found to be expressed in higher levels in many tumours such as glioma, colorectal adenoma, hepatoma, non-small lung cancer, retinoblastoma, melanoma and cervical cancer [30–35]. Musashi-1 maintains proliferation of gastric carcinoma cells as evidenced by its positive correlation with PCNA [36]. Musashi-1 controls the Notch signaling cascade by inhibiting the activity of its repressor m-Numb. Interestingly, p21WAF1 is also found to be a target of Musashi-1 [37, 38]. Musashi-1 plays a significant role in several neurodegenerative disorders including Alzheimer’s disease and epilepsy [31,
39]. Particularly, Musashi regulates insulin expression and proliferation in diabetes as a consequence of endoplasmic reticulum-related stress [40]. Recently, studies reveal a stimulatory role of Musashi-1 in inducing WNT pathway through the activation of Frat-1. Musashi-1 induces the proliferation of mammary gland cells and regulates the growth of colon carcinoma. Notably, Musashi-1 maintains progenitor cell homeostasis by regulating Wnt, Notch and thyroid pathways which are affected during colon tumourigenesis [41]. Musashi-1 is also found to be expressed in cancer stem cells. Additionally, the knockdown of Musashi-1 results in decreased proliferation, growth arrest and apoptosis in a number of tumours including colon adenocarcinoma [42, 43]. Moreover, Musashi-1 has been found to be highly expressed in preneoplastic and neoplastic tissues of oral squamous cell carcinoma [7]. In our previous study, we have investigated the expression pattern of Musashi-1 in preneoplastic and neoplastic oral squamous cell carcinoma tissues. However, the prognostic significance of Musashi-1 was uncovered in oral cancer. To our knowledge, the expression of Nestin has not been demonstrated in the multistep process of oral carcinogenesis. Moreover, its prognostic significance was not studied extensively in oral cancer. Based on this background, we have examined the expression pattern of Nestin in the progressive stages of oral squamous cell carcinoma. The association between Nestin and Musashi-1 expression was also analysed in oral carcinoma tissues. Furthermore, the prognostic significance of both Musashi-1 and Nestin has been investigated to explore the possibility of using both markers for worst prognosis and therapeutic purposes.
Materials and methods Tissue samples Tissue samples from precancerous lesions and carcinoma [mild–moderate dysplasia (36), severe dysplasia (28) and oral squamous cell carcinoma (72)] were obtained from the Royapettah Hospital, Chennai. In addition, we have collected 15 normal samples from patients undergoing orthodontic surgery. The samples have been fixed immediately with 10 % buffered formalin and they were processed for paraffin embedding. The sections were cut at 4 μm and mounted on coated glass slides. The histological grading of dysplasia and oral squamous cell carcinoma have been assessed according to the criteria illustrated by Pindborg et al. [44] by two pathologists until a consensus was reached. The range of follow-up is 12–50 months with a mean follow-up period of 31.7. The clearance was obtained from the Hospital Medical Board with permission from the Directorate of Medical Education, Government of Tamil Nadu, India (No. 21823/ME 1/1/2008).
Clin Oral Invest
Immunohistochemistry
Table 1 Expression of Nestin in precancerous and cancerous tissues of oral squamous epithelium
Immunohistochemistry was done on tissue sections as described previously [7]. The sections were deparaffinized in xylene and rehydrated through graded alcohols, and the antigen retrieval was done by heating the slides in 0.1 M sodium citrate buffer (pH 6.0) in a microwave oven. After cooling, the endogenous peroxidase activity was blocked by placing the slides in 3 % hydrogen peroxide for 10 min. The non-specific binding sites were blocked with 0.3 % bovine serum albumin (BSA) for 30 min at room temperature. The sections were then incubated (overnight at 4 °C) with mouse monoclonal antiMusashi-1 (Santa Cruz Biotechnology, USA) and goat polyclonal anti-Nestin (Santa Cruz Biotechnology, USA) in a dilution of 1:200 for the above-mentioned antibodies. The slides were immunostained using the respective horseradish peroxidase-conjugated secondary antibodies (Invitrogen, USA). The chromogen 3,3′-diaminobenzidine was then used as a substrate for localizing the antibody binding. After counterstaining with haematoxylin, the sections were mounted and viewed under a microscope. Negative controls without primary antibody were included for each staining. The immunostained sections were scored according to the percentage of stained cells in more than three random areas of cancer tissue as examined in ×200 magnification. Two independent observers without prior knowledge on the patient’s clinicopathological data assessed the immunostained slides. The staining was assessed as 0 (negative), no staining or staining in less than 5 % of cells; 1+ (mild), staining in 5–15 % of carcinoma cells; 2+ (moderate), staining in 16–25 % of carcinoma cells; and 3+ (intense), staining in >25 % of carcinoma
No. of patients
Fig. 1 Immunostaining of Nestin in the normal (a), mild dysplasia (b), severe dysplasia (c) and carcinoma (d) (inset: cytoplasmic staining of Musashi-1 in oral carcinoma cells) tissues of oral squamous epithelium. Bar= 100 μm
Nestin 0
1+
2+
3+
Normal
15
13
2
0
0
Mild–moderate Severe Carcinoma
36 28 72
11 4 7
14 7 13
9 8 22
2 9 30a
0 (negative), no staining or 25 % positive cells a
Expression of Nestin shows a significant difference between different groups calculated by chi-square (p
