Clin Transl Oncol DOI 10.1007/s12094-014-1173-y
BRIEF RESEARCH ARTICLE
Aldehyde dehydrogenases in early stage lung cancer: nuclear expression A. Lo´pez-Gonza´lez • C. Salas • M. Provencio M. Co´rdoba • C. Gamallo
•
Received: 17 November 2013 / Accepted: 21 February 2014 Ó Federacio´n de Sociedades Espan˜olas de Oncologı´a (FESEO) 2014
Abstract Purpose Aldehyde dehydrogenase enzymes are a family of intracellular enzymes that participate in cellular detoxification, differentiation and drug resistance through the oxidation of cellular aldehydes. The isoform 1 (ALDH1) has been proved useful for the identification of cancer stem cells. The ALDH1 cytoplasmatic expression has been associated with poor prognostis in several tumours, such as non-small cell lung cancer. The role of the ALDH1 nuclear expression remains unknown. Methods We conducted a historical cohort study in 89 patients diagnosed of stage I non-small cell lung cancer treated with surgery between 2009 and 2004 in the Thoracic Surgery Department in the Universitary Hospital Puerta de Hierro. We selected from this sample those cases with nuclear expression of the ALDH1.
A. Lo´pez-Gonza´lez Medical Oncology, Complejo Asistencial Universitario Leo´n, Leo´n, Spain e-mail: [email protected] C. Salas Pathological Anatomy, Hospital Universitario Puerta de Hierro, Majadahonda, Spain M. Provencio Medical Oncology, Hospital Universitario Puerta de Hierro, Majadahonda, Spain M. Co´rdoba Thoracic Surgery Department, Hospital Universitario Puerta de Hierro, Majadahonda, Spain C. Gamallo (&) Pathological Anatomy, Hospital Universitario La Princesa, Madrid, Spain e-mail: [email protected]
Results Three of the 89 (3.3 %) patients showed a nuclear expression of the ALDH1. The three of them are still alive with a median time of follow up of 73 months (more than 6 years). Conclusion We have identified ALDH1 as a nuclear protein in early stage non-small cell lung cancer. It might have a function in cell cycle control, associating a better prognosis to these patients. More studies are necessary to clarify the role of nuclear expression of ALDH1. Keywords Aldehyde dehydrogenase Nuclear expression Lung cancer
Introduction Non-small cell lung cancer is the most frequent tumour in the world, with 1,400,000 newly diagnosed patients in a year. The 55 % of the cases are diagnosed in an advanced stage, a 30 % with regional node invasion and only a 15 % in an early stage. In this last group, surgery is the treatment of choice. More than 50 % of the patients die in the first 5 years [1]. 50 % of the recurrences are diagnosed in the first 24 months after the curative treatment, and more than 90 % in the first 5 years [2]. We need to identify early stage lung cancer patients at high risk for recurrence, who would then benefit most from adjuvant therapies. The cancer stem cell hypothesis for the origins of tumour self-renewal has received special attention in recent times. According to the hypothesis, cancers arise from stem cells that have acquired sufficient oncogenic mutations for transformation. Therefore, the tumour cell of origin, referred to as a tumour-initiating cell, is a stem cell that is capable of self-renewal and differentiation [3]. The existence of lung cancer stem cells could
123
Clin Transl Oncol
Fig. 1 Nuclear expression of the ALDH1 in the epidermoid (case 1), adenocarcinoma (case 2) and mucoepidermoid tumor (case 3)
explain why current treatments for lung cancer cannot eradicate tumour cells and have reached a therapeutic plateau, because these therapies target the bulk of cancer and are unlikely to eliminate cancer stem cells (CSC) [4]. And thus, residual lung CSC may regenerate a cancer cell population, leading to tumour relapse after therapy.
123
The ALDH family is a cytosolic isoenzyme responsible for oxidizing intracellular aldehydes to carboxylic acid. Nineteen different isoforms have been described [5]. The ALDH1 can have a role in the initial differentiation of the stem cell, through the oxidation of retinol in retinoic acid [6]. Recently, several articles have described the cytoplasmatic expression of ALDH1 as an unfavourable
Clin Transl Oncol
prognostic factor in epithelial cancers, such as breast [7, 8], lung [4, 9] or bladder [10]. The role of the nuclear expression of ALDH1 is unknown. It has been suggested as a regulator of the cell cycle [11]. We describe here three patients diagnosed of an early stage non-small cell lung cancer and nuclear expression of ALDH1 with no evidence of recurrence of disease after more than 5 years.
Materials and methods We conducted a historical cohort study in 89 patients diagnosed of stage I non-small cell lung cancer treated with surgery between 2009 and 2004 in the Thoracic Surgery Department in the Universitary Hospital Puerta de Hierro. They received no adjuvant chemotherapy treatment. The paraffin tissue samples were cut with a rotary microtome. First of all, they are initially dewaxed in xylol, re-hydrated in decreasing gradation alcohols and subsequently washed in PBS (phosphate buffered saline). Then, the endogenous peroxidise block is performed and antigen retrieval is processed by a pressure cooker for 2 min in EDTA at pH 8. Then, they are incubated for 1 h with ALDH1 specific antibody (Abcam) dilution 1/100. The complex Envision?peroxidase is used as visualization system. The product of the antigen–antibody reaction is developed with a diaminobenzidine solution and H202. Nucleus was stained with Harris hematoxylin (15 s) and the samples were dehydrated in alcohols of increasing concentration to finally being mounted on a permanent medium (Eukitt) (O. Kindler and Co; GMBGH Freiburg, Germany). We have selected from this sample those cases with nuclear expression of the ALDH1.
carcinoma, one mucoepidermoid and one adenocarcinoma (Fig. 1). The three of them are still alive with a median time of follow up of 73 months (more than 6 years). Data from the three cases are shown in Table 1. A cytoplasmatic expression of the ALDH1 is shown in Fig. 2.
Discussion It is thought that basal cells of the lung have the capacity for multi-potent differentiation as well as self-renewal. The histologically different tumour types in the lung come from the accumulation of oncogenic mutations. Patel et al. [12] described in 2008 the evidence for ALDH as a relevant lung cancer stem cell marker. One year later, the ALDH1 cytoplasmatic expression was correlated with poor prognosis [4]. The nuclear expression of ALDH1 has never been described in lung tumour, neither in other tumours. So far, the only nuclear expression of ALDH described to our knowledge is in the cornea [13]. At this level, the cytoplasmatic expression of ALDH family plays important roles, such as vision, embryonic development and neurotransmission. They also protect the eye from UV-induced damage and may contribute to a structural role in the cornea.
Results Three of the 89 (3.3 %) patients showed a nuclear expression of the ALDH1. The three patients were male, with a median age of 56 years. The history of smoking was very heterogenous, from non-smokers to current smokers. There is also disparity in histology, with one squamous
Fig. 2 Cytoplasmatic expression of ALDH1
Table 1 Summary of the three patients with nuclear expression of ALDH1 Sex
Age
Smoking
Histology
Current status
Time of follow-up (months)
Patient 1
Male
66 years
Ex-smoker
Adenocarcinoma
AWD
63
Patient 2
Male
31 years
Non-smoker
Mucoepidermoid
AWD
61
Patient 3
Male
73 years
Current smoker
Squamous carcinoma
AWD
96
AWD alive without disease
123
Clin Transl Oncol
The hypothesis of ALDH as a nuclear factor of mitotic control becomes important after the observation of an inverse relationship between ALDH expression and cell proliferation rate. It is observed in the cornea that a highly proliferating epithelium is associated with a minor ALDH gene expression, and that when ALDH is transfected, the corneal epithelium shows reduced growth rate and prolonged cell cycle length [14]. Although our sample is small with only three patients, it is remarkable that all of them are alive after more than 6 years of median follow-up. It is also important that neither smoking nor histology are factors in common between them. Thus, we believe that nuclear expression of ALDH might play an important role in survival.
Conclusion Nuclear expression of ALDH in early stage of NSCLC might have a function in cell cycle control, associating a better prognosis to these patients. More studies are necessary to clarify the role of nuclear expression of ALDH1. Conflict of interest
None.
References 1. Mountain CF. A new international staging system for lung cancer. Chest. 1986;89:225–33.
123
2. Pairolero PC, Williams DE, Bergstralh EJ, Piehler JM, Bernatz PE, Payne WS. Postsurgical stage I bronchogenic carcinoma: morbid implications of recurrent disease. Ann Thorac Surg. 1984;38:331–6. 3. Sullivan J, Minna JD, Shay JW. Evidence for self-renewing lung cancer stem cells and their implications in tumor iniaition, progresio´n, and targeted therapy. Cancer Metastasis Rev. 2010;29:61–72. 4. Jiang F, Qiu Q, Khanna A, Todd NW, Deepak J, Xing L, et al. Aldehyde dehydrogenase 1 is a tumor stem cell-associated marker in lung cancer. Mol Cancer Res. 2009;7:330–8. 5. Douville J, Beaulieu R, Balicki D. ALDH1 as a functional marker of cancer item and progenitor cells. Stem Cells Dev. 2009;18(1):17–25. 6. Yoshida A, Hsu LC, Dave´ V. Retinal oxidation activity and biological role of human cytosolic aldehyde dehydrogenase. Enzyme. 1992;46:239–44. 7. Morimoto K, Kim SJ, Tanei T, Shimazu K, Tanji Y, Taguchi T, et al. Stem cell marker aldehyde dehydrogenase 1-positive breast cancer are characterized by negative estro´geno receptor, positive human epidermal growth factor receptor type 2, and high Ki67 expression. Cancer Sci. 2009;100:1062–8. 8. Charape-Jauffret E, Ginestier C, Iovino F, Tarpin C, Diebel M, Esterni B, et al. Aldehyde dehydrogenase 1 positive cancer item cells mediate metastasis and poor clinical outcome in inflammatory breast cancer. Clin Cancer Res. 2010;16:45–55. 9. Shien K, Toyooka S, Ichimura K, Soh J, Furukawa M, Maki Y, et al. Prognostic impact of cancer stem cell-related markers in non-small cell lung cancer patients treated with induction chemoradiotherapy. Lung Cancer. 2012;77: 162–7. 10. Su Y, Qiu Q, Zhang X, Jiang Z, Leng Q, Liu Z, et al. Aldehyde dehydrogenase 1-positive cell population is enriched in tumor-initiating cells and associated with progresio´n of bladder cancer. Cancer Epidemiol Biomark Prev. 2010;19:327–37. 11. Stagos D, Chen Y, Cantore M, Jester JV, Vasiliou V. Corneal aldehyde dehydrogenases: multiple functions and novel nuclear localization. Brain Res Bull. 2010;15:211–8. 12. Patel M, Lu L, Zander DS, Sreerama L, Coco D, Moreb JS. ALDH1A1 and ALDH3A1 expression in lung cancers: correlation with histologic type and potential precursors. Lung Cancer. 2008;59(3):340–9. 13. Stagos D, Lu L, Zander DS, Sreerama L, Coco D, Moreb JS. Corneal aldehyde dehydrogenases: multiple functions and novel nuclear localization. Brain Res Bull. 2010;15:21–218. 14. Pappa A, Brown D, Koutalos Y, DeGregori J, White C, Vasiliou V. Human aldehyde dehydrogenase 3A1 inhibits proliferation and promotes survival of human corneal epitelial cells. J Biol Chem. 2005;280:27998–8006.
BRIEF RESEARCH ARTICLE
Aldehyde dehydrogenases in early stage lung cancer: nuclear expression A. Lo´pez-Gonza´lez • C. Salas • M. Provencio M. Co´rdoba • C. Gamallo
•
Received: 17 November 2013 / Accepted: 21 February 2014 Ó Federacio´n de Sociedades Espan˜olas de Oncologı´a (FESEO) 2014
Abstract Purpose Aldehyde dehydrogenase enzymes are a family of intracellular enzymes that participate in cellular detoxification, differentiation and drug resistance through the oxidation of cellular aldehydes. The isoform 1 (ALDH1) has been proved useful for the identification of cancer stem cells. The ALDH1 cytoplasmatic expression has been associated with poor prognostis in several tumours, such as non-small cell lung cancer. The role of the ALDH1 nuclear expression remains unknown. Methods We conducted a historical cohort study in 89 patients diagnosed of stage I non-small cell lung cancer treated with surgery between 2009 and 2004 in the Thoracic Surgery Department in the Universitary Hospital Puerta de Hierro. We selected from this sample those cases with nuclear expression of the ALDH1.
A. Lo´pez-Gonza´lez Medical Oncology, Complejo Asistencial Universitario Leo´n, Leo´n, Spain e-mail: [email protected] C. Salas Pathological Anatomy, Hospital Universitario Puerta de Hierro, Majadahonda, Spain M. Provencio Medical Oncology, Hospital Universitario Puerta de Hierro, Majadahonda, Spain M. Co´rdoba Thoracic Surgery Department, Hospital Universitario Puerta de Hierro, Majadahonda, Spain C. Gamallo (&) Pathological Anatomy, Hospital Universitario La Princesa, Madrid, Spain e-mail: [email protected]
Results Three of the 89 (3.3 %) patients showed a nuclear expression of the ALDH1. The three of them are still alive with a median time of follow up of 73 months (more than 6 years). Conclusion We have identified ALDH1 as a nuclear protein in early stage non-small cell lung cancer. It might have a function in cell cycle control, associating a better prognosis to these patients. More studies are necessary to clarify the role of nuclear expression of ALDH1. Keywords Aldehyde dehydrogenase Nuclear expression Lung cancer
Introduction Non-small cell lung cancer is the most frequent tumour in the world, with 1,400,000 newly diagnosed patients in a year. The 55 % of the cases are diagnosed in an advanced stage, a 30 % with regional node invasion and only a 15 % in an early stage. In this last group, surgery is the treatment of choice. More than 50 % of the patients die in the first 5 years [1]. 50 % of the recurrences are diagnosed in the first 24 months after the curative treatment, and more than 90 % in the first 5 years [2]. We need to identify early stage lung cancer patients at high risk for recurrence, who would then benefit most from adjuvant therapies. The cancer stem cell hypothesis for the origins of tumour self-renewal has received special attention in recent times. According to the hypothesis, cancers arise from stem cells that have acquired sufficient oncogenic mutations for transformation. Therefore, the tumour cell of origin, referred to as a tumour-initiating cell, is a stem cell that is capable of self-renewal and differentiation [3]. The existence of lung cancer stem cells could
123
Clin Transl Oncol
Fig. 1 Nuclear expression of the ALDH1 in the epidermoid (case 1), adenocarcinoma (case 2) and mucoepidermoid tumor (case 3)
explain why current treatments for lung cancer cannot eradicate tumour cells and have reached a therapeutic plateau, because these therapies target the bulk of cancer and are unlikely to eliminate cancer stem cells (CSC) [4]. And thus, residual lung CSC may regenerate a cancer cell population, leading to tumour relapse after therapy.
123
The ALDH family is a cytosolic isoenzyme responsible for oxidizing intracellular aldehydes to carboxylic acid. Nineteen different isoforms have been described [5]. The ALDH1 can have a role in the initial differentiation of the stem cell, through the oxidation of retinol in retinoic acid [6]. Recently, several articles have described the cytoplasmatic expression of ALDH1 as an unfavourable
Clin Transl Oncol
prognostic factor in epithelial cancers, such as breast [7, 8], lung [4, 9] or bladder [10]. The role of the nuclear expression of ALDH1 is unknown. It has been suggested as a regulator of the cell cycle [11]. We describe here three patients diagnosed of an early stage non-small cell lung cancer and nuclear expression of ALDH1 with no evidence of recurrence of disease after more than 5 years.
Materials and methods We conducted a historical cohort study in 89 patients diagnosed of stage I non-small cell lung cancer treated with surgery between 2009 and 2004 in the Thoracic Surgery Department in the Universitary Hospital Puerta de Hierro. They received no adjuvant chemotherapy treatment. The paraffin tissue samples were cut with a rotary microtome. First of all, they are initially dewaxed in xylol, re-hydrated in decreasing gradation alcohols and subsequently washed in PBS (phosphate buffered saline). Then, the endogenous peroxidise block is performed and antigen retrieval is processed by a pressure cooker for 2 min in EDTA at pH 8. Then, they are incubated for 1 h with ALDH1 specific antibody (Abcam) dilution 1/100. The complex Envision?peroxidase is used as visualization system. The product of the antigen–antibody reaction is developed with a diaminobenzidine solution and H202. Nucleus was stained with Harris hematoxylin (15 s) and the samples were dehydrated in alcohols of increasing concentration to finally being mounted on a permanent medium (Eukitt) (O. Kindler and Co; GMBGH Freiburg, Germany). We have selected from this sample those cases with nuclear expression of the ALDH1.
carcinoma, one mucoepidermoid and one adenocarcinoma (Fig. 1). The three of them are still alive with a median time of follow up of 73 months (more than 6 years). Data from the three cases are shown in Table 1. A cytoplasmatic expression of the ALDH1 is shown in Fig. 2.
Discussion It is thought that basal cells of the lung have the capacity for multi-potent differentiation as well as self-renewal. The histologically different tumour types in the lung come from the accumulation of oncogenic mutations. Patel et al. [12] described in 2008 the evidence for ALDH as a relevant lung cancer stem cell marker. One year later, the ALDH1 cytoplasmatic expression was correlated with poor prognosis [4]. The nuclear expression of ALDH1 has never been described in lung tumour, neither in other tumours. So far, the only nuclear expression of ALDH described to our knowledge is in the cornea [13]. At this level, the cytoplasmatic expression of ALDH family plays important roles, such as vision, embryonic development and neurotransmission. They also protect the eye from UV-induced damage and may contribute to a structural role in the cornea.
Results Three of the 89 (3.3 %) patients showed a nuclear expression of the ALDH1. The three patients were male, with a median age of 56 years. The history of smoking was very heterogenous, from non-smokers to current smokers. There is also disparity in histology, with one squamous
Fig. 2 Cytoplasmatic expression of ALDH1
Table 1 Summary of the three patients with nuclear expression of ALDH1 Sex
Age
Smoking
Histology
Current status
Time of follow-up (months)
Patient 1
Male
66 years
Ex-smoker
Adenocarcinoma
AWD
63
Patient 2
Male
31 years
Non-smoker
Mucoepidermoid
AWD
61
Patient 3
Male
73 years
Current smoker
Squamous carcinoma
AWD
96
AWD alive without disease
123
Clin Transl Oncol
The hypothesis of ALDH as a nuclear factor of mitotic control becomes important after the observation of an inverse relationship between ALDH expression and cell proliferation rate. It is observed in the cornea that a highly proliferating epithelium is associated with a minor ALDH gene expression, and that when ALDH is transfected, the corneal epithelium shows reduced growth rate and prolonged cell cycle length [14]. Although our sample is small with only three patients, it is remarkable that all of them are alive after more than 6 years of median follow-up. It is also important that neither smoking nor histology are factors in common between them. Thus, we believe that nuclear expression of ALDH might play an important role in survival.
Conclusion Nuclear expression of ALDH in early stage of NSCLC might have a function in cell cycle control, associating a better prognosis to these patients. More studies are necessary to clarify the role of nuclear expression of ALDH1. Conflict of interest
None.
References 1. Mountain CF. A new international staging system for lung cancer. Chest. 1986;89:225–33.
123
2. Pairolero PC, Williams DE, Bergstralh EJ, Piehler JM, Bernatz PE, Payne WS. Postsurgical stage I bronchogenic carcinoma: morbid implications of recurrent disease. Ann Thorac Surg. 1984;38:331–6. 3. Sullivan J, Minna JD, Shay JW. Evidence for self-renewing lung cancer stem cells and their implications in tumor iniaition, progresio´n, and targeted therapy. Cancer Metastasis Rev. 2010;29:61–72. 4. Jiang F, Qiu Q, Khanna A, Todd NW, Deepak J, Xing L, et al. Aldehyde dehydrogenase 1 is a tumor stem cell-associated marker in lung cancer. Mol Cancer Res. 2009;7:330–8. 5. Douville J, Beaulieu R, Balicki D. ALDH1 as a functional marker of cancer item and progenitor cells. Stem Cells Dev. 2009;18(1):17–25. 6. Yoshida A, Hsu LC, Dave´ V. Retinal oxidation activity and biological role of human cytosolic aldehyde dehydrogenase. Enzyme. 1992;46:239–44. 7. Morimoto K, Kim SJ, Tanei T, Shimazu K, Tanji Y, Taguchi T, et al. Stem cell marker aldehyde dehydrogenase 1-positive breast cancer are characterized by negative estro´geno receptor, positive human epidermal growth factor receptor type 2, and high Ki67 expression. Cancer Sci. 2009;100:1062–8. 8. Charape-Jauffret E, Ginestier C, Iovino F, Tarpin C, Diebel M, Esterni B, et al. Aldehyde dehydrogenase 1 positive cancer item cells mediate metastasis and poor clinical outcome in inflammatory breast cancer. Clin Cancer Res. 2010;16:45–55. 9. Shien K, Toyooka S, Ichimura K, Soh J, Furukawa M, Maki Y, et al. Prognostic impact of cancer stem cell-related markers in non-small cell lung cancer patients treated with induction chemoradiotherapy. Lung Cancer. 2012;77: 162–7. 10. Su Y, Qiu Q, Zhang X, Jiang Z, Leng Q, Liu Z, et al. Aldehyde dehydrogenase 1-positive cell population is enriched in tumor-initiating cells and associated with progresio´n of bladder cancer. Cancer Epidemiol Biomark Prev. 2010;19:327–37. 11. Stagos D, Chen Y, Cantore M, Jester JV, Vasiliou V. Corneal aldehyde dehydrogenases: multiple functions and novel nuclear localization. Brain Res Bull. 2010;15:211–8. 12. Patel M, Lu L, Zander DS, Sreerama L, Coco D, Moreb JS. ALDH1A1 and ALDH3A1 expression in lung cancers: correlation with histologic type and potential precursors. Lung Cancer. 2008;59(3):340–9. 13. Stagos D, Lu L, Zander DS, Sreerama L, Coco D, Moreb JS. Corneal aldehyde dehydrogenases: multiple functions and novel nuclear localization. Brain Res Bull. 2010;15:21–218. 14. Pappa A, Brown D, Koutalos Y, DeGregori J, White C, Vasiliou V. Human aldehyde dehydrogenase 3A1 inhibits proliferation and promotes survival of human corneal epitelial cells. J Biol Chem. 2005;280:27998–8006.
