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Immunohistochemical analysis of aldehyde dehydrogenase isoforms and their association with estrogen-receptor status and disease progression in breast cancer

Immunohistochemical analysis of aldehyde dehydrogenase isoforms and their association with... Breast Cancer: Targets and Therapy Dovepress open access to scientific and medical research open Access Full Text Article Sho r T r epo r T Immunohistochemical analysis of aldehyde dehydrogenase isoforms and their association with estrogen-receptor status and disease progression in breast cancer 1,2 Lynn M opdenaker Abstract: In many types of tumors, especially breast tumors, aldehyde dehydrogenase (ALDH) 1,3 activity has been used to identify cancer stem-like cells within the tumor. The presence and Kimberly M Arnold 3,4 quantity of these cells are believed to predict the response of tumors to chemotherapy. Therefore, ryan T pohlig 1 identification and eradication of these cells would be necessary to cure the patient. However, Jayasree S padmanabhan there are 19 different ALDH isoforms that could contribute to the enzyme activity. ALDH1A1 1,3 Daniel C Flynn and ALDH1A3 are among the isoforms mostly responsible for the increased ALDH activity 1–3 Jennifer Sims-Mourtada observed in these stem-like cells, although the main isoforms vary in different tissues and Center for Translational Cancer tumor types. In the study reported here, we attempted to determine if ALDH1A1 or ALDH1A3, r esearch, h elen F Graham Cancer specifically, correlate with tumor stage, grade, and hormone-receptor status in breast-cancer Center, Christiana Care h ealth Services, Inc., Newark, Delaware, USA; patients. While there was no significant correlation between ALDH1A1 and any of the parameters Department of Biological Sciences, tested, we were able to identify a positive correlation between ALDH1A3 and tumor stage in Department of Medical Laborator y triple-negative cancers. In addition, ALDH1A3 was negatively correlated with estrogen-receptor Sciences, Biostatistics Core Facility, University of Delaware, Newark, status. Our data suggest that ALDH1A3 could be utilized as a marker to identify stem-like cells Delaware, USA within triple-negative tumors. Keywords: breast tumor, ALDH, ALDH1A1, ALDH1A3, stem-like cells, triple-negative cancer Introduction The aldehyde dehydrogenase (ALDH) family of enzymes are NAD(P)+-dependent enzymes that metabolize aromatic and aliphatic aldehydes. They are involved in oxi- dation of all-trans-retinal and 9-cis-retinal, which function in the retinoic acid (RA) cell-signaling pathway, and regulate cellular differentiation, cell-cycle progression, and apoptosis. High ALDH activity has been associated with self-renewal in a variety of normal and tumor tissues including those of the prostate, breast, lung, colon, cervix, and 2–6 ovary. Recently, ALDH activity has become a functional marker for breast-cancer stem-like cells. When breast-cancer cells are stratified using the Aldefluor assay, Correspondence: Jennifer Sims-Mourtada which measures the activity of ALDH using a fluorescent substrate, ALDH bright Center for Translational Cancer (ALDHbr) cells represent the tumor-initiating population, and are associated with research, helen F Graham Cancer 7,8 Center and research Institute drug resistance and metastasis. The presence of ALDHbr cells in the residual tumors Christiana Care health Services, Inc., of women who failed neoadjuvant chemotherapy predicted worse overall survival, 4701 ogletown Stanton rd Suite 4300, Newark, Delaware 19713, USA indicating that these cells represent a resistant stem-like population. Tel +1 302 623 4648 There are 19 mammalian ALDH genes, belonging to eleven families and four sub- Fax +1 302 623 4314 1,10–12 email jsimsmourtada@christianacare.org families, whose expression and enzyme level vary among tissues. Of these, only a submit your manuscript | www.dovepress.com Breast Cancer: Targets and Therapy 2014:6 205–209 Dovepress © 2014 Opdenaker et al. This work is published by Dove Medical Press Limited, and licensed under Creative Commons Attribution – Non Commercial (unported, v3.0) License. The full terms of the License are available at http://creativecommons.org/licenses/by-nc/3.0/. Non-commercial uses of the work are permitted without any further http://dx.doi.org/10.2147/BCTT.S73674 permission from Dove Medical Press Limited, provided the work is properly attributed. Permissions beyond the scope of the License are administered by Dove Medical Press Limited. Information on how to request permission may be found at: http://www.dovepress.com/permissions.php opdenaker et al Dovepress few have been shown to function in the RA signaling pathway. receptor 2 (HER2) expression were determined. Hematoxylin These include members of the ALDH1 family (ALDH1A1, and eosin stains of tumor sections were reviewed by a breast- ALDH1A3) as well as ALDH8A1. Immunohistochemical cancer pathologist to determine the percentage of tumor studies using pan-ALDH1 antibodies have shown that over- nuclei and necrosis. expression of ALDH1 in breast-cancer specimens is predic- tive of worse overall survival and is most often observed in Immunohistochemical procedure 13–15 aggressive tumors. Whole 4 µm tissue sections of each specimen were analyzed The isoforms which contribute to the high functional by immunohistochemical staining using the LSAB+ System- activity in the Aldefluor assay are likely to be tissue specific. HRP staining kit (Dako) according to the manufacturer’s In normal breast tissue, the predominant isoform responsible instructions. Antibodies to ALDH1A1 (clone EP1933Y, for RA signaling is ALDH1A1 and it was predicted that, Abcam) and ALDH1A3 (cat number ab80176, Abcam) were like many other tissues, ALDH1A1 activity was responsible used at a 1:100 dilution for staining. Specificity of staining for ALDHbr cells. However, in breast-cancer cell lines, was confirmed by omission of the primary antibody and ALDH1A3 has been shown to be the dominant isoform. staining with an isotype-matched control antibody (Jackson Marcato et al published that knocking down ALDH1A1 only Laboratories). Human liver sections served as a positive minimally affected the Aldeu fl or activity in breast cells, while control for staining. Slides were scored as having no expres- the number of ALDHbr cells was significantly reduced with sion (0), or weak (1), moderate (2), or strong (3) tumor-cell knockdown of ALDH1A3. They further demonstrated that staining. protein expression of ALDH1A3, but not that of ALDH1A1, correlated with Aldefluor activity in breast-cancer cell lines Statistical analysis and patient specimens. Two sequential regression models were used to test the Expression of ALDH1A1 has been associated with relationship between being triple negative (TN) and stain- estrogen-receptor (ER) positivity in luminal progenitors dur- ing for ALDH1A1 and ALDH1A3 after adjusting for grade ing normal breast development. Likewise, estrogen signal- and stage. Assumptions for the regression analysis were ing has been shown to decrease expression of ALDH1A3 in met (residual normality, multicollinearity, linearity, and female breast tissue. The relationship between the expres- homoscedasticity). With the relatively small sample size sion of ALDH1 isoforms and ER expression in breast cancer it was not possible to run analysis by the full spectrum of is unclear. In malignant breast tissue, overexpression of both stages (Table 1); therefore, stage was dichotomized by group- ALDH1A1 and ALDH1A3 has been reported, however, only ing 1A, 1B, and 2A together and 2B through 4 in a second ALDH1A3 is reported to be positively associated with tumor group. The Spearman correlation matrices for the TN and grade, stage, and metastasis. In this report, we report on the ER-positive groups are presented in Table 2. Spearman was expression of ALDH1A1 and ALDH1A3 in breast-cancer used because normality was not satisfied for every relation - subtypes and determine their association to receptor status, ship using a bivariate analysis. grade, and progression. Results Methods patient demographics patient population This study comprised a cohort of 62 patients with invasive Breast-cancer tissue specimens were obtained from the ductal carcinoma. Of these, 25 were ER positive and 37 biobank at the Helen F Graham Cancer Center and Research lacked expression of ER, progesterone receptor, or HER2 Institute under an institutional review board-approved (TN). A summary of the clinical data and demographics protocol. The patient population consisted of women who from the patient cohort is provided in Table 1. Tumor sections underwent surgery at the institute between the years 2006 from 31 individuals with early stage breast cancer and from and 2013 and gave informed written consent for the samples 26 with advanced stage (clinical stage $2B) were included to be biobanked. Upon surgical resection, tumors were pre- in the analysis. Serial sections were immunolabeled for pared as routine formalin-fixed, paraffin-embedded blocks expression of ALDH1A1 or ALDH1A3. Slides were scored for sectioning. Pathological examination was performed for as having no expression (0), weak (1), moderate (2) or, strong patient care, clinicopathologic details were recorded, and ER, (3) tumor-cell staining (Figure 1 and Table 1). Although progesterone receptor, and human epidermal growth factor slight stromal staining was observed in several sections for submit your manuscript | www.dovepress.com Breast Cancer: Targets and Therapy 2014:6 Dovepress Dovepress Aldehyde dehydrogenase isoforms and breast cancer Table 1 patient demographics and isoform immunolabeling Table 2 Spearman’s correlation matrix intensities Tumor Estrogen-receptor positive properties Tumor properties TN Total ER+ Grade BMI ALDH1A1 ALDH1A3 Stage n % n % n % Grade 0.172 0.288 0.522 –0.024 P-value 0.455 0.194 0.013 0.916 pr status BMI 0.154 –0.161 –0.112 0.204 positive 17 68 0 0 17 27 P-value 0.583 0.474 0.621 0.362 Negative 8 32 37 100 45 73 oneA1 0.577 0.188 -0.176 -0.018 Stage P-value 0.312 0.944 0.003 0.369 1A 2 8 4 11 6 19 oneA3 0.264 0.384 1B 0 0 1 3 1 3 -0.034 -0.284 2A 12 48 12 32 24 80 P-value 0.847 0.27 0.114 0.058 2B 8 32 4 11 12 43 Stage 0.211 -0.075 0.221 0.536 3A 2 8 4 11 6 19 P-value 0.264 0.774 0.223 0.002 3B 0 0 0 0 0 0 Triple negative 3C 1 4 5 14 6 18 Note: Bold values indicate significance. 4 0 0 2 5 2 5 Abbreviation: BMI, body mass index. Grade 1 4 18 0 0 4 7 2 11 50 7 20 18 32 ciation was found between the immunolabeling intensity of 3 7 32 28 80 35 61 ALDH1A1, receptor status, grade, and stage. The base model race consisting of dichotomized stage and grade was not signic fi ant White/Caucasian 20 80 7 28 27 108 2 2 African American 3 12 20 80 23 92 (F(2,50) =1.65, P=0.20, R =0.06, adjusted R =0.03), nor were Asian 2 8 0 0 2 8 grade or stage individually signic fi ant. After adjusting for Unknown 0 0 8 32 8 32 grade and stage, there was no signic fi ant association between ALDh1A1 receptor status and ALDH1A1 expression (F(3,49) =1.64, 0 10 40 4 11 14 23 2 2 2 1 6 24 14 38 20 32 P=0.19, R =0.09, adjusted R =0.04, ∆R =0.03). Independent 2 6 24 13 35 19 31 of ALDH1A1 expression, receptor expression (b=0.39, SE 3 3 12 6 16 9 15 =0.32), grade (b=0.12, SE =0.25), and stage (b=0.33, SE ALDh1A3 =0.27) were not associated (all P-values .0.05). 0 11 44 3 8 14 23 1 9 36 7 19 16 26 2 3 12 16 43 19 31 Analysis of ALDh1A3 expression 3 2 8 11 30 13 21 Analysis of ALDH1A3 staining in the all breast-cancer Abbreviations: er+, estrogen-receptor positive; pr, progesterone receptor; TN, specimens revealed no staining in 23% (14 of 62), expression triple negative. was weak in 26% (16 of 62), moderate in 31% (19 of 62), and strong in 21% (13 of 62) of samples (Table 1). Of the both antibodies, this staining was not included in the analysis ER-positive samples, 44% (eleven of 25) had no expression as stromal staining of ALDH1 has been reported in normal 20 of ALDH1A3, 36% (nine of 25) had weak expression, 12% breast stroma. (three of 25) had moderate expression, and 8% (two of 25) Analysis of ALDh1A1 expression had strong expression. For TN samples, 8% (three of 37) The distribution of staining by specimen type is presented in had no expression of ALDH1A3. Expression was weak in Table 1. Of all of the breast-cancer samples, 23% (14 of 62) 19% (seven of 37), moderate in 43% (16 of 37), and strong had no expression of ALDH1A1, 32% (20 of 62) had weak in 30% (eleven of 37) of samples. In contrast to ALDH1A1 expression, 31% (19 of 62) had moderate expression, and expression, an association of ALDH1A3 to tumor grade and 15% (nine of 62) had strong expression. Samples were further stage was observed for ER-positive tumors. The base model categorized by receptor status. Of the ER-positive tumors, consisting of dichotomized stage and grade was significant 2 2 40% (ten of 25) had no expression of ALDH1A1, 24% had (F(2,50) =10.90, P,0.001, R =0.3, adjusted R =0.28). Both weak (six of 25) expression, 24% (six of 25) had moderate grade (P=0.002) and stage (P=0.003) were also individually expression, and 12% (three of 25) had strong expression. For significant. Furthermore, in contrast to ALDH1A1, expres - TN samples, 11% (four of 37) had no expression, 38% (14 of sion of ALDH1A3 was associated with hormone-receptor 37) had weak expression, 35% (13 of 37) had moderate expres- status. After adjusting for grade and stage, TN specimens sion, and 16% (six of 37) had strong expression. No asso- had a signic fi antly stronger association with ALDH1A3 than submit your manuscript | www.dovepress.com Breast Cancer: Targets and Therapy 2014:6 Dovepress opdenaker et al Dovepress Figure 1 Immunohistochemical analysis of ALDh1A1 and ALDh1A3 in breast-cancer specimens. representative images are shown of negative, weak, moderate, and strong immunolabeling. Note: Magnification is 20 ×. ER-positive samples (F(3,49) =12.29, P,0.001, R =0.43, However, in contrast to our findings here, they did not find 2 2 adjusted R =0.39, ∆R =0.13). In this model, TN receptor a correlation between ALDH1A3 and ER status. Although status (b=0.89, SE =0.27, t(49) =3.29, P=0.002) and higher our findings indicate that ALDH1A3 is associated with TN stage (b=0.79, SE =0.23, t(49) =3.47, P=0.001), were both tumors, a further categorization of ER- tumors may be needed positively related to ALDH1A3. In contrast to ER-positive to clarify this relationship. TN breast cancers can be further tumors, there was no association between ALDH1A3 and classified by gene-expression profiles such as basal-like/ grade within the TN subtype (b=0.28, SE =0.21, t(49) =1.33, claudin-low, mesenchymal stem-like, and luminal androgen P=0.19). This is most likely due to the fact that the majority receptor subtypes. The basal-like subtype is defined by the of the TN samples presented as high grade. expression of cytokeratins 5 and 14 and the epidermal growth factor receptor (EGFR). Basal-like tumors often have a poor Discussion prognosis and are reported to have a higher percentage of cells In this study, we sought to examine the expression of ALDH1A that possess a stem-like phenotype. Recent studies have family members ALDH1A1 and ALDH1A3 in breast cancer shown that expression of ALDH1A correlates with prognosis 15,26 in relation to stage, grade, and hormone-receptor expression. in EGFR-expressing basal-like cancers. However, specic fi We observed that both isoforms were significantly expressed isoforms were not examined in these studies. in both ER-positive and TN tumors. However, there was no Our findings indicate that ALDH1A3 correlates with significant association between immunolabeling intensity of stage in TN breast-cancer samples, and may be a prognos- ALDH1A1 and tumor stage or receptor status. In contrast, tic marker for TN breast cancer. Limitations of this study ALDH1A3 was expressed at signic fi antly higher levels in include small sample size, and lack of progression-free and tumors that lacked expression of the ER. In both ER and TN overall-survival information. Additionally, the TN samples tumors, expression of ALDH1A3 was positively correlated in this study were not further classified into basal-like and with tumor stage, and there was a positive association with luminal subtypes. Future work should examine the relation- grade in ER-positive tumors. Our studies support previous ship of ALDH1A3 and survival within the subtypes of TN n fi dings that indicate the expression of ALDH1A3 is inversely breast cancer. Additionally, the relationship between ALDH 18,19 correlated with ER signaling, and may be predictive of isoforms and other prognostic markers, such as Ki67, and metastatic potential in invasive breast cancers. EGFR should be determined. To date, there have been many conflicting reports regard - ing expression of ALDH1A isoforms and their prognostic Disclosure value. Some groups have found that ALDH1A is a marker of The authors declare no conflicts of interest in this work. 8,21 poor clinical outcome and others have found no correlation 17,22,23 between ALDH1A expression and clinical outcome. References Analysis of ALDH isoforms has shown that only ALDH1A3 1. Black W, Vasiliou V. The aldehyde dehydrogenase gene superfamily resource center. Hum Genomics. 2009;4(2):136–142. correlates with distant metastases, disease-free survival, and 2. Marcato P, Dean CA, Giacomantonio CA, Lee PW. Aldehyde overall survival in breast cancer. Similarly, Qiu et al found dehydrogenase: its role as a cancer stem cell marker comes down to the a correlation between ALDH1A3 and poor prognosis. specific isoform. Cell Cycle. 2011;10(9):1378–1384. submit your manuscript | www.dovepress.com Breast Cancer: Targets and Therapy 2014:6 Dovepress Dovepress Aldehyde dehydrogenase isoforms and breast cancer 3. Liu SY, Zheng PS. High aldehyde dehydrogenase activity identifies 15. Morimoto K, Kim SJ, Tanei T, et al. Stem cell marker aldehyde dehydro- cancer stem cells in human cervical cancer. Oncotarget. 2013;4(12): genase 1-positive breast cancers are characterized by negative estrogen 2462–2475. receptor, positive human epidermal growth factor receptor type 2, and 4. Ucar D, Cogle CR, Zucali JR, et al. Aldehyde dehydrogenase activity as high Ki67 expression. Cancer Sci. 2009;100(6):1062–1068. a functional marker for lung cancer. Chem Biol Interact. 2009;178(1–3): 16. Petrosino JM, Disilvestro D, Ziouzenkova O. 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Primary breast cancer patients with 19. Yasmeen R, Reichert B, Deiuliis J, et al. Autocrine function of aldehyde high risk clinicopathologic features have high percentages of bone dehydrogenase 1 as a determinant of diet- and sex-specific differences marrow epithelial cells with ALDH activity and CD44 CD24lo cancer in visceral adiposity. Diabetes. 2013;62(1):124–136. stem cell phenotype. Eur J Cancer. 2011;47(10):1527–1536. 20. Ali HR, Dawson SJ, Blows FM, Provenzano E, Pharoah PD, Caldas C. 8. Ginestier C, Hur MH, Charafe-Jauffret E, et al. ALDH1 is a marker of Cancer stem cell markers in breast cancer: pathological, clinical and normal and malignant human mammary stem cells and a predictor of prognostic significance. Breast Cancer Res. 2011;13(6):R118. poor clinical outcome. Cell Stem Cell. 2007;1(5):555–567. 21. Charafe-Jauffret E, Ginestier C, Iovino F, et al. Aldehyde dehydroge- 9. Alamgeer M, Ganju V, Kumar B, et al. Changes in aldehyde dehydroge- nase 1-positive cancer stem cells mediate metastasis and poor clinical nase-1 expression during neoadjuvant chemotherapy predict outcome in outcome in inflammatory breast cancer. Clin Cancer Res. 2010;16(1): locally advanced breast cancer. Breast Cancer Res. 2014;16(2): R44. 45–55. 10. Marchitti SA, Brocker C, Stagos D, Vasiliou V. Non-P450 aldehyde 22. Neumeister V, Agarwal S, Bordeaux J, Camp RL, Rimm DL. In situ oxidizing enzymes: the aldehyde dehydrogenase superfamily. Expert identification of putative cancer stem cells by multiplexing ALDH1, Opin Drug Metab Toxicol. 2008;4(6):697–720. CD44, and cytokeratin identifies breast cancer patients with poor 11. Vasiliou V, Nebert DW. Analysis and update of the human aldehyde prognosis. Am J Pathol. 2010;176(5):2131–2138. dehydrogenase (ALDH) gene family. Hum Genomics. 2005;2(2): 23. Resetkova E, Reis-Filho JS, Jain RK, et al. 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J Clin Pathol. 2011;64(11):937–946. 14. Currie MJ, Beardsley BE, Harris GC, et al. Immunohistochemical analysis of cancer stem cell markers in invasive breast carcinoma and associated ductal carcinoma in situ: relationships with markers of tumor hypoxia and microvascularity. Hum Pathol. 2012;44(3):402–411. Breast Cancer: Targets and Therapy Dovepress Publish your work in this journal Breast Cancer: Targets and Therapy is an international, peer- View the full aims and scopes of this journal here. The manuscript reviewed open access journal focusing on breast cancer research, management system is completely online and includes a very quick identification of therapeutic targets and the optimal use of preven - and fair peer-review system, which is all easy to use. 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Immunohistochemical analysis of aldehyde dehydrogenase isoforms and their association with estrogen-receptor status and disease progression in breast cancer

Breast Cancer : Targets and Therapy , Volume 6 – Dec 12, 2014

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Abstract

Breast Cancer: Targets and Therapy Dovepress open access to scientific and medical research open Access Full Text Article Sho r T r epo r T Immunohistochemical analysis of aldehyde dehydrogenase isoforms and their association with estrogen-receptor status and disease progression in breast cancer 1,2 Lynn M opdenaker Abstract: In many types of tumors, especially breast tumors, aldehyde dehydrogenase (ALDH) 1,3 activity has been used to identify cancer stem-like cells within the tumor. The presence and Kimberly M Arnold 3,4 quantity of these cells are believed to predict the response of tumors to chemotherapy. Therefore, ryan T pohlig 1 identification and eradication of these cells would be necessary to cure the patient. However, Jayasree S padmanabhan there are 19 different ALDH isoforms that could contribute to the enzyme activity. ALDH1A1 1,3 Daniel C Flynn and ALDH1A3 are among the isoforms mostly responsible for the increased ALDH activity 1–3 Jennifer Sims-Mourtada observed in these stem-like cells, although the main isoforms vary in different tissues and Center for Translational Cancer tumor types. In the study reported here, we attempted to determine if ALDH1A1 or ALDH1A3, r esearch, h elen F Graham Cancer specifically, correlate with tumor stage, grade, and hormone-receptor status in breast-cancer Center, Christiana Care h ealth Services, Inc., Newark, Delaware, USA; patients. While there was no significant correlation between ALDH1A1 and any of the parameters Department of Biological Sciences, tested, we were able to identify a positive correlation between ALDH1A3 and tumor stage in Department of Medical Laborator y triple-negative cancers. In addition, ALDH1A3 was negatively correlated with estrogen-receptor Sciences, Biostatistics Core Facility, University of Delaware, Newark, status. Our data suggest that ALDH1A3 could be utilized as a marker to identify stem-like cells Delaware, USA within triple-negative tumors. Keywords: breast tumor, ALDH, ALDH1A1, ALDH1A3, stem-like cells, triple-negative cancer Introduction The aldehyde dehydrogenase (ALDH) family of enzymes are NAD(P)+-dependent enzymes that metabolize aromatic and aliphatic aldehydes. They are involved in oxi- dation of all-trans-retinal and 9-cis-retinal, which function in the retinoic acid (RA) cell-signaling pathway, and regulate cellular differentiation, cell-cycle progression, and apoptosis. High ALDH activity has been associated with self-renewal in a variety of normal and tumor tissues including those of the prostate, breast, lung, colon, cervix, and 2–6 ovary. Recently, ALDH activity has become a functional marker for breast-cancer stem-like cells. When breast-cancer cells are stratified using the Aldefluor assay, Correspondence: Jennifer Sims-Mourtada which measures the activity of ALDH using a fluorescent substrate, ALDH bright Center for Translational Cancer (ALDHbr) cells represent the tumor-initiating population, and are associated with research, helen F Graham Cancer 7,8 Center and research Institute drug resistance and metastasis. The presence of ALDHbr cells in the residual tumors Christiana Care health Services, Inc., of women who failed neoadjuvant chemotherapy predicted worse overall survival, 4701 ogletown Stanton rd Suite 4300, Newark, Delaware 19713, USA indicating that these cells represent a resistant stem-like population. Tel +1 302 623 4648 There are 19 mammalian ALDH genes, belonging to eleven families and four sub- Fax +1 302 623 4314 1,10–12 email jsimsmourtada@christianacare.org families, whose expression and enzyme level vary among tissues. Of these, only a submit your manuscript | www.dovepress.com Breast Cancer: Targets and Therapy 2014:6 205–209 Dovepress © 2014 Opdenaker et al. This work is published by Dove Medical Press Limited, and licensed under Creative Commons Attribution – Non Commercial (unported, v3.0) License. The full terms of the License are available at http://creativecommons.org/licenses/by-nc/3.0/. Non-commercial uses of the work are permitted without any further http://dx.doi.org/10.2147/BCTT.S73674 permission from Dove Medical Press Limited, provided the work is properly attributed. Permissions beyond the scope of the License are administered by Dove Medical Press Limited. Information on how to request permission may be found at: http://www.dovepress.com/permissions.php opdenaker et al Dovepress few have been shown to function in the RA signaling pathway. receptor 2 (HER2) expression were determined. Hematoxylin These include members of the ALDH1 family (ALDH1A1, and eosin stains of tumor sections were reviewed by a breast- ALDH1A3) as well as ALDH8A1. Immunohistochemical cancer pathologist to determine the percentage of tumor studies using pan-ALDH1 antibodies have shown that over- nuclei and necrosis. expression of ALDH1 in breast-cancer specimens is predic- tive of worse overall survival and is most often observed in Immunohistochemical procedure 13–15 aggressive tumors. Whole 4 µm tissue sections of each specimen were analyzed The isoforms which contribute to the high functional by immunohistochemical staining using the LSAB+ System- activity in the Aldefluor assay are likely to be tissue specific. HRP staining kit (Dako) according to the manufacturer’s In normal breast tissue, the predominant isoform responsible instructions. Antibodies to ALDH1A1 (clone EP1933Y, for RA signaling is ALDH1A1 and it was predicted that, Abcam) and ALDH1A3 (cat number ab80176, Abcam) were like many other tissues, ALDH1A1 activity was responsible used at a 1:100 dilution for staining. Specificity of staining for ALDHbr cells. However, in breast-cancer cell lines, was confirmed by omission of the primary antibody and ALDH1A3 has been shown to be the dominant isoform. staining with an isotype-matched control antibody (Jackson Marcato et al published that knocking down ALDH1A1 only Laboratories). Human liver sections served as a positive minimally affected the Aldeu fl or activity in breast cells, while control for staining. Slides were scored as having no expres- the number of ALDHbr cells was significantly reduced with sion (0), or weak (1), moderate (2), or strong (3) tumor-cell knockdown of ALDH1A3. They further demonstrated that staining. protein expression of ALDH1A3, but not that of ALDH1A1, correlated with Aldefluor activity in breast-cancer cell lines Statistical analysis and patient specimens. Two sequential regression models were used to test the Expression of ALDH1A1 has been associated with relationship between being triple negative (TN) and stain- estrogen-receptor (ER) positivity in luminal progenitors dur- ing for ALDH1A1 and ALDH1A3 after adjusting for grade ing normal breast development. Likewise, estrogen signal- and stage. Assumptions for the regression analysis were ing has been shown to decrease expression of ALDH1A3 in met (residual normality, multicollinearity, linearity, and female breast tissue. The relationship between the expres- homoscedasticity). With the relatively small sample size sion of ALDH1 isoforms and ER expression in breast cancer it was not possible to run analysis by the full spectrum of is unclear. In malignant breast tissue, overexpression of both stages (Table 1); therefore, stage was dichotomized by group- ALDH1A1 and ALDH1A3 has been reported, however, only ing 1A, 1B, and 2A together and 2B through 4 in a second ALDH1A3 is reported to be positively associated with tumor group. The Spearman correlation matrices for the TN and grade, stage, and metastasis. In this report, we report on the ER-positive groups are presented in Table 2. Spearman was expression of ALDH1A1 and ALDH1A3 in breast-cancer used because normality was not satisfied for every relation - subtypes and determine their association to receptor status, ship using a bivariate analysis. grade, and progression. Results Methods patient demographics patient population This study comprised a cohort of 62 patients with invasive Breast-cancer tissue specimens were obtained from the ductal carcinoma. Of these, 25 were ER positive and 37 biobank at the Helen F Graham Cancer Center and Research lacked expression of ER, progesterone receptor, or HER2 Institute under an institutional review board-approved (TN). A summary of the clinical data and demographics protocol. The patient population consisted of women who from the patient cohort is provided in Table 1. Tumor sections underwent surgery at the institute between the years 2006 from 31 individuals with early stage breast cancer and from and 2013 and gave informed written consent for the samples 26 with advanced stage (clinical stage $2B) were included to be biobanked. Upon surgical resection, tumors were pre- in the analysis. Serial sections were immunolabeled for pared as routine formalin-fixed, paraffin-embedded blocks expression of ALDH1A1 or ALDH1A3. Slides were scored for sectioning. Pathological examination was performed for as having no expression (0), weak (1), moderate (2) or, strong patient care, clinicopathologic details were recorded, and ER, (3) tumor-cell staining (Figure 1 and Table 1). Although progesterone receptor, and human epidermal growth factor slight stromal staining was observed in several sections for submit your manuscript | www.dovepress.com Breast Cancer: Targets and Therapy 2014:6 Dovepress Dovepress Aldehyde dehydrogenase isoforms and breast cancer Table 1 patient demographics and isoform immunolabeling Table 2 Spearman’s correlation matrix intensities Tumor Estrogen-receptor positive properties Tumor properties TN Total ER+ Grade BMI ALDH1A1 ALDH1A3 Stage n % n % n % Grade 0.172 0.288 0.522 –0.024 P-value 0.455 0.194 0.013 0.916 pr status BMI 0.154 –0.161 –0.112 0.204 positive 17 68 0 0 17 27 P-value 0.583 0.474 0.621 0.362 Negative 8 32 37 100 45 73 oneA1 0.577 0.188 -0.176 -0.018 Stage P-value 0.312 0.944 0.003 0.369 1A 2 8 4 11 6 19 oneA3 0.264 0.384 1B 0 0 1 3 1 3 -0.034 -0.284 2A 12 48 12 32 24 80 P-value 0.847 0.27 0.114 0.058 2B 8 32 4 11 12 43 Stage 0.211 -0.075 0.221 0.536 3A 2 8 4 11 6 19 P-value 0.264 0.774 0.223 0.002 3B 0 0 0 0 0 0 Triple negative 3C 1 4 5 14 6 18 Note: Bold values indicate significance. 4 0 0 2 5 2 5 Abbreviation: BMI, body mass index. Grade 1 4 18 0 0 4 7 2 11 50 7 20 18 32 ciation was found between the immunolabeling intensity of 3 7 32 28 80 35 61 ALDH1A1, receptor status, grade, and stage. The base model race consisting of dichotomized stage and grade was not signic fi ant White/Caucasian 20 80 7 28 27 108 2 2 African American 3 12 20 80 23 92 (F(2,50) =1.65, P=0.20, R =0.06, adjusted R =0.03), nor were Asian 2 8 0 0 2 8 grade or stage individually signic fi ant. After adjusting for Unknown 0 0 8 32 8 32 grade and stage, there was no signic fi ant association between ALDh1A1 receptor status and ALDH1A1 expression (F(3,49) =1.64, 0 10 40 4 11 14 23 2 2 2 1 6 24 14 38 20 32 P=0.19, R =0.09, adjusted R =0.04, ∆R =0.03). Independent 2 6 24 13 35 19 31 of ALDH1A1 expression, receptor expression (b=0.39, SE 3 3 12 6 16 9 15 =0.32), grade (b=0.12, SE =0.25), and stage (b=0.33, SE ALDh1A3 =0.27) were not associated (all P-values .0.05). 0 11 44 3 8 14 23 1 9 36 7 19 16 26 2 3 12 16 43 19 31 Analysis of ALDh1A3 expression 3 2 8 11 30 13 21 Analysis of ALDH1A3 staining in the all breast-cancer Abbreviations: er+, estrogen-receptor positive; pr, progesterone receptor; TN, specimens revealed no staining in 23% (14 of 62), expression triple negative. was weak in 26% (16 of 62), moderate in 31% (19 of 62), and strong in 21% (13 of 62) of samples (Table 1). Of the both antibodies, this staining was not included in the analysis ER-positive samples, 44% (eleven of 25) had no expression as stromal staining of ALDH1 has been reported in normal 20 of ALDH1A3, 36% (nine of 25) had weak expression, 12% breast stroma. (three of 25) had moderate expression, and 8% (two of 25) Analysis of ALDh1A1 expression had strong expression. For TN samples, 8% (three of 37) The distribution of staining by specimen type is presented in had no expression of ALDH1A3. Expression was weak in Table 1. Of all of the breast-cancer samples, 23% (14 of 62) 19% (seven of 37), moderate in 43% (16 of 37), and strong had no expression of ALDH1A1, 32% (20 of 62) had weak in 30% (eleven of 37) of samples. In contrast to ALDH1A1 expression, 31% (19 of 62) had moderate expression, and expression, an association of ALDH1A3 to tumor grade and 15% (nine of 62) had strong expression. Samples were further stage was observed for ER-positive tumors. The base model categorized by receptor status. Of the ER-positive tumors, consisting of dichotomized stage and grade was significant 2 2 40% (ten of 25) had no expression of ALDH1A1, 24% had (F(2,50) =10.90, P,0.001, R =0.3, adjusted R =0.28). Both weak (six of 25) expression, 24% (six of 25) had moderate grade (P=0.002) and stage (P=0.003) were also individually expression, and 12% (three of 25) had strong expression. For significant. Furthermore, in contrast to ALDH1A1, expres - TN samples, 11% (four of 37) had no expression, 38% (14 of sion of ALDH1A3 was associated with hormone-receptor 37) had weak expression, 35% (13 of 37) had moderate expres- status. After adjusting for grade and stage, TN specimens sion, and 16% (six of 37) had strong expression. No asso- had a signic fi antly stronger association with ALDH1A3 than submit your manuscript | www.dovepress.com Breast Cancer: Targets and Therapy 2014:6 Dovepress opdenaker et al Dovepress Figure 1 Immunohistochemical analysis of ALDh1A1 and ALDh1A3 in breast-cancer specimens. representative images are shown of negative, weak, moderate, and strong immunolabeling. Note: Magnification is 20 ×. ER-positive samples (F(3,49) =12.29, P,0.001, R =0.43, However, in contrast to our findings here, they did not find 2 2 adjusted R =0.39, ∆R =0.13). In this model, TN receptor a correlation between ALDH1A3 and ER status. Although status (b=0.89, SE =0.27, t(49) =3.29, P=0.002) and higher our findings indicate that ALDH1A3 is associated with TN stage (b=0.79, SE =0.23, t(49) =3.47, P=0.001), were both tumors, a further categorization of ER- tumors may be needed positively related to ALDH1A3. In contrast to ER-positive to clarify this relationship. TN breast cancers can be further tumors, there was no association between ALDH1A3 and classified by gene-expression profiles such as basal-like/ grade within the TN subtype (b=0.28, SE =0.21, t(49) =1.33, claudin-low, mesenchymal stem-like, and luminal androgen P=0.19). This is most likely due to the fact that the majority receptor subtypes. The basal-like subtype is defined by the of the TN samples presented as high grade. expression of cytokeratins 5 and 14 and the epidermal growth factor receptor (EGFR). Basal-like tumors often have a poor Discussion prognosis and are reported to have a higher percentage of cells In this study, we sought to examine the expression of ALDH1A that possess a stem-like phenotype. Recent studies have family members ALDH1A1 and ALDH1A3 in breast cancer shown that expression of ALDH1A correlates with prognosis 15,26 in relation to stage, grade, and hormone-receptor expression. in EGFR-expressing basal-like cancers. However, specic fi We observed that both isoforms were significantly expressed isoforms were not examined in these studies. in both ER-positive and TN tumors. However, there was no Our findings indicate that ALDH1A3 correlates with significant association between immunolabeling intensity of stage in TN breast-cancer samples, and may be a prognos- ALDH1A1 and tumor stage or receptor status. In contrast, tic marker for TN breast cancer. Limitations of this study ALDH1A3 was expressed at signic fi antly higher levels in include small sample size, and lack of progression-free and tumors that lacked expression of the ER. In both ER and TN overall-survival information. Additionally, the TN samples tumors, expression of ALDH1A3 was positively correlated in this study were not further classified into basal-like and with tumor stage, and there was a positive association with luminal subtypes. Future work should examine the relation- grade in ER-positive tumors. Our studies support previous ship of ALDH1A3 and survival within the subtypes of TN n fi dings that indicate the expression of ALDH1A3 is inversely breast cancer. Additionally, the relationship between ALDH 18,19 correlated with ER signaling, and may be predictive of isoforms and other prognostic markers, such as Ki67, and metastatic potential in invasive breast cancers. EGFR should be determined. To date, there have been many conflicting reports regard - ing expression of ALDH1A isoforms and their prognostic Disclosure value. Some groups have found that ALDH1A is a marker of The authors declare no conflicts of interest in this work. 8,21 poor clinical outcome and others have found no correlation 17,22,23 between ALDH1A expression and clinical outcome. References Analysis of ALDH isoforms has shown that only ALDH1A3 1. Black W, Vasiliou V. The aldehyde dehydrogenase gene superfamily resource center. Hum Genomics. 2009;4(2):136–142. correlates with distant metastases, disease-free survival, and 2. Marcato P, Dean CA, Giacomantonio CA, Lee PW. Aldehyde overall survival in breast cancer. 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