www.nature.com/scientificreports
OPEN
received: 09 February 2016 accepted: 27 July 2016 Published: 25 August 2016
Prognostic significance of nuclear expression of UMP-CMP kinase in triple negative breast cancer patients Ning Qing Liu1,2,∗,†, Tommaso De Marchi1,2,∗, Annemieke Timmermans1, Anita M. A. C. Trapman-Jansen1, Renée Foekens1, Maxime P. Look1, Marcel Smid1, Carolien H. M. van Deurzen3, Paul N. Span4, Fred C. G. J. Sweep5, Julie Benedicte Brask6, Vera Timmermans-Wielenga6, John A. Foekens1, John W. M. Martens1 & Arzu Umar1 We have previously identified UMP-CMP kinase (CMPK1) as a prognostic marker for triple negative breast cancer (TNBC) by mass spectrometry (MS). In this study we evaluated CMPK1 association to prognosis in an independent set of samples by immunohistochemistry (IHC) and assessed biological pathways associated to its expression through gene set enrichment analysis (GSEA). A total of 461 TNBC paraffin-embedded tissues were collected from different academic hospitals in Europe, incorporated into tissue micro-arrays (TMA), and stained for CMPK1 expression. We also collected gene expression data of 60 samples, which were also present in the TMA, for GSEA correlation analysis. CMPK1 IHC staining showed both cytoplasmic and nuclear components. While cytoplasmic CMPK1 did not show any association to metastasis free survival (MFS), nuclear CMPK1 was associated to poor prognosis independently from other prognostic factors in stratified Cox regression analyses. GSEA correlation analysis of the nuclear CMPK1-stratified gene expression dataset showed a significant enrichment of extracellular matrix (ECM; positive correlation) and cell cycle (negative correlation) associated genes. We have shown here that nuclear CMPK1 is indicative of poor prognosis in TNBCs and that its expression may be related to dysregulation of ECM and cell cycle molecules. TNBC is a subtype of breast cancer that lacks expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (Her2)1. Patients diagnosed with TNBC generally manifest adverse clinical outcome when compared to other breast cancer subtypes and, to date, there is no clinically available targeted therapy for these patients2. In this perspective, the majority of TNBC patients receive adjuvant chemotherapy after resection of the primary tumor. Recently, we identified an 11-protein signature that predicts prognosis of TNBC patients by using laser capture microdissection coupled to high resolution MS analysis3. While this signature displayed high sensitivity and specificity for predicting disease outcome, introduction of a proteomic workflow as the one used to develop our classifier4 into a clinical setting is not yet feasible due to extensive sample preparation and measurement times. In this perspective, an immuno-based (e.g. IHC) assay would constitute a suitable approach to application in clinical diagnostics. CMPK1, as part of the 11-protein signature, was identified as a prognostic marker for lymph-node negative (LNN) and adjuvant systemic chemotherapy naive 1
Department of Medical Oncology, Erasmus MC Cancer Institute, Erasmus University Medical Center, Rotterdam, The Netherlands. 2Postgraduate School of Molecular Medicine, Erasmus University Medical Center, Rotterdam, The Netherlands. 3Department of Pathology, Erasmus MC Cancer Institute, Erasmus University Medical Center, Rotterdam, The Netherlands. 4Department of Radiation Oncology, Radboud University Medical Center, Nijmegen, The Netherlands. 5Department of Laboratory Medicine, Radboud University Medical Center, Nijmegen, The Netherlands. 6Department of Pathology, Centre of Diagnostic Investigations, Copenhagen University Hospital, Copenhagen, Denmark. †Present address: Department of Molecular Biology, Faculty of Science, Nijmegen Centre for Molecular Life Sciences, Radboud University, Nijmegen, The Netherlands. ∗These authors contributed equally to this work. Correspondence and requests for materials should be addressed to N.Q.L. (email: [email protected]) or T.D.M. (email: [email protected]) Scientific Reports | 6:32027 | DOI: 10.1038/srep32027
1
www.nature.com/scientificreports/
Figure 1. Schematic representation of experimental workflow. (ASCN) TNBC patients. In the current study, we used a TMA platform to further investigate the clinical significance of CMPK1 in TNBC patients. CMPK1 is a 22 kDa enzyme that catalyzes phosphorylation of pyrimidine nucleoside monophosphates, which is essential for de novo biosynthesis of pyrimidines5,6. Also, CMPK1 performs a crucial role in activation of nucleoside analogues used as chemotherapy against human cancers and pathogenic viruses7. In a neoplastic setting, decreased expression of CMPK1 mRNA has been associated with 5-fluorouracil resistance of a colorectal cancer cell line HCT-88. Some genetic polymorphisms of CMPK1 have also been reported as a prognostic marker for non-small cell lung cancer9 and pancreatic cancer10 patients treated with a gemcitabine-based chemotherapy. CMPK1 has been observed in different subcellular locations of HeLa S3 cancer cells, as it predominantly localizes in the cytoplasm but can also enter the nucleus6. Subcellular localization of this protein may be important for cancer progression, but no study has yet revealed whether this has prognostic or predictive value in breast cancer. Therefore, further investigation may help to understand the clinical value of this marker, including its subcellular localization. In this study, we evaluated whether CMPK1 expression in different subcellular compartments is associated to patient prognosis in TNBC patients by IHC analysis of TMA derived from different academic medical centers in Europe. In order to elucidate the molecular pathways associated to subcellular localization of CMPK1, we performed GSEA correlation analysis on a subset of tumors incorporated in the TMA, of which gene expression data was available. A schematic representation of this workflow is shown in Fig. 1.
Results
Expression and subcellular localization of CMPK1 in triple negative breast cancer tissues.
Analysis of the staining results of an anti-CMPK1 antibody on breast tissues showed that CMPK1 prevalently stained epithelial cells of the mammary gland (normal cells: Fig. S1; carcinoma cells: Fig. S2A–D). When analyzing breast carcinoma tissues, both nuclear and cytoplasmic localization of CMPK1 protein was observed, as previously described in cancer cell lines6. Cytoplasmic CMPK1 (cCMPK1) was generally ubiquitous, as the majority of tissues (80.2%) expressed cCMPK1 in more (>) than 70% of carcinoma. Regarding intensity distribution, only a small percentage (9.5%) of tissues was found negative or showed a faint staining (7.0%), while most tissues displayed either weak (33.3%) or moderate (35.5%) cCMPK1 staining. Only 14.7% of cores showed strong cCMPK1 staining intensity (Fig. S2E–F). On the other hand, nuclear CMPK1 (nCMPK1) staining showed differences in terms of quantity of stained carcinoma cells and staining intensity. Nearly a third (29.7%) of cores did not show any nCMPK1 staining, while positive tissues displayed large heterogeneity in the number of stained tumor cells: 10.6% of tissues showed staining in 1–10% of tumor cells, while 12.8% and 17.2% displayed a number of stained carcinoma cells between 11–25% and 26–50%, respectively. One third (i.e. 29.7%) of tissue cores showed nCMPK1 staining in more than 50% of carcinoma cells (8.8% and 20.9% of the tumors showed expression in 51–70% and >70% of the tumor cells, respectively; Fig. S2E). When assessing nCMPK1 staining intensity distribution, the majority of positive tissues showed weak (16.5%), moderate (23.4%) and strong (24.5%) stainings, with only a small percentage (5.9%) showing faint staining (Fig. S2F). Overall these data show that, while cCMPK1 presents a generally ubiquitous and homogeneous staining, nCMPK1 staining varies widely in terms of stained tumor cells and staining intensity.
Differences between the TMA datasets. We next compared the three TMA datasets with respect to possible differences in clinical and histopathological parameters (Table 1). The RUMC and CUH cohorts comprised Scientific Reports | 6:32027 | DOI: 10.1038/srep32027
2
www.nature.com/scientificreports/ Total n of patients
RUMC*
CUH*
EMC*
Total*
48 (100.0)
83 (100.0)
267 (100.0)
398 (100.0)
Age (years) ≤40
7 (14.6)
12 (14.5)
65 (24.3)
84 (21.1)
41–55
18 (37.5)
22 (26.5)
113 (42.3)
153 (38.4)
56–70
19 (39.6)
29 (34.9)
67 (25.1)
115 (28.9)
4 (8.3)
20 (24.1)
22 (8.3)
46 (11.6)
>70 Menopausal status Premenopausal
18 (37.5)
24 (28.9)
155 (58.0)
197 (49.5)
Postmenopausal
30 (62.5)
59 (71.1)
112 (42.0)
201 (50.5)
Tumor size pT1
22 (45.8)
49 (59.1)
107 (40.1)
178 (44.7)
pT2 + pTx
24 (50.0)
30 (36.1)
147 (55.0)
201 (50.5)
pT3 + pT4
2 (4.2)
4 (4.8)
13 (4.9)
19 (4.8)
Good
0 (0.0)
2 (2.4)
4 (1.5)
6 (1.5)
Moderate
5 (10.4)
8 (9.6)
44 (16.5)
57 (14.3)
Poor
32 (66.7)
54 (65.1)
213 (79.8)
299 (75.1)
Unknown
11 (22.9)
19 (22.9)
6 (2.2)
36 (9.1)
Tumor Grade
Lymph node positivity 46 (95.8)
83 (100.0)
202 (75.6)
331 (83.2)
1 to 3
0
2 (4.2)
0 (0.0)
44 (16.5)
46 (11.6)
>3
0 (0.0)
0 (0.0)
21 (7.9)
21 (5.2)
Adjuvant chemotherapy Yes
2 (4.2)
40 (48.2)
83 (31.1)
125 (31.4)
No
46 (95.8)
43 (51.8)
184 (68.9)
273 (68.6)
Table 1. Clinical and histopathological characteristics of patients included in the TMAs. *Data are displayed as number (percentage). Acronyms: CUH: Copenhagen University Hospital; EMC: Erasmus University Medical Center; RUMC: Radboud University Medical Center.
a higher number of postmenopausal women (χ2 test P
OPEN
received: 09 February 2016 accepted: 27 July 2016 Published: 25 August 2016
Prognostic significance of nuclear expression of UMP-CMP kinase in triple negative breast cancer patients Ning Qing Liu1,2,∗,†, Tommaso De Marchi1,2,∗, Annemieke Timmermans1, Anita M. A. C. Trapman-Jansen1, Renée Foekens1, Maxime P. Look1, Marcel Smid1, Carolien H. M. van Deurzen3, Paul N. Span4, Fred C. G. J. Sweep5, Julie Benedicte Brask6, Vera Timmermans-Wielenga6, John A. Foekens1, John W. M. Martens1 & Arzu Umar1 We have previously identified UMP-CMP kinase (CMPK1) as a prognostic marker for triple negative breast cancer (TNBC) by mass spectrometry (MS). In this study we evaluated CMPK1 association to prognosis in an independent set of samples by immunohistochemistry (IHC) and assessed biological pathways associated to its expression through gene set enrichment analysis (GSEA). A total of 461 TNBC paraffin-embedded tissues were collected from different academic hospitals in Europe, incorporated into tissue micro-arrays (TMA), and stained for CMPK1 expression. We also collected gene expression data of 60 samples, which were also present in the TMA, for GSEA correlation analysis. CMPK1 IHC staining showed both cytoplasmic and nuclear components. While cytoplasmic CMPK1 did not show any association to metastasis free survival (MFS), nuclear CMPK1 was associated to poor prognosis independently from other prognostic factors in stratified Cox regression analyses. GSEA correlation analysis of the nuclear CMPK1-stratified gene expression dataset showed a significant enrichment of extracellular matrix (ECM; positive correlation) and cell cycle (negative correlation) associated genes. We have shown here that nuclear CMPK1 is indicative of poor prognosis in TNBCs and that its expression may be related to dysregulation of ECM and cell cycle molecules. TNBC is a subtype of breast cancer that lacks expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (Her2)1. Patients diagnosed with TNBC generally manifest adverse clinical outcome when compared to other breast cancer subtypes and, to date, there is no clinically available targeted therapy for these patients2. In this perspective, the majority of TNBC patients receive adjuvant chemotherapy after resection of the primary tumor. Recently, we identified an 11-protein signature that predicts prognosis of TNBC patients by using laser capture microdissection coupled to high resolution MS analysis3. While this signature displayed high sensitivity and specificity for predicting disease outcome, introduction of a proteomic workflow as the one used to develop our classifier4 into a clinical setting is not yet feasible due to extensive sample preparation and measurement times. In this perspective, an immuno-based (e.g. IHC) assay would constitute a suitable approach to application in clinical diagnostics. CMPK1, as part of the 11-protein signature, was identified as a prognostic marker for lymph-node negative (LNN) and adjuvant systemic chemotherapy naive 1
Department of Medical Oncology, Erasmus MC Cancer Institute, Erasmus University Medical Center, Rotterdam, The Netherlands. 2Postgraduate School of Molecular Medicine, Erasmus University Medical Center, Rotterdam, The Netherlands. 3Department of Pathology, Erasmus MC Cancer Institute, Erasmus University Medical Center, Rotterdam, The Netherlands. 4Department of Radiation Oncology, Radboud University Medical Center, Nijmegen, The Netherlands. 5Department of Laboratory Medicine, Radboud University Medical Center, Nijmegen, The Netherlands. 6Department of Pathology, Centre of Diagnostic Investigations, Copenhagen University Hospital, Copenhagen, Denmark. †Present address: Department of Molecular Biology, Faculty of Science, Nijmegen Centre for Molecular Life Sciences, Radboud University, Nijmegen, The Netherlands. ∗These authors contributed equally to this work. Correspondence and requests for materials should be addressed to N.Q.L. (email: [email protected]) or T.D.M. (email: [email protected]) Scientific Reports | 6:32027 | DOI: 10.1038/srep32027
1
www.nature.com/scientificreports/
Figure 1. Schematic representation of experimental workflow. (ASCN) TNBC patients. In the current study, we used a TMA platform to further investigate the clinical significance of CMPK1 in TNBC patients. CMPK1 is a 22 kDa enzyme that catalyzes phosphorylation of pyrimidine nucleoside monophosphates, which is essential for de novo biosynthesis of pyrimidines5,6. Also, CMPK1 performs a crucial role in activation of nucleoside analogues used as chemotherapy against human cancers and pathogenic viruses7. In a neoplastic setting, decreased expression of CMPK1 mRNA has been associated with 5-fluorouracil resistance of a colorectal cancer cell line HCT-88. Some genetic polymorphisms of CMPK1 have also been reported as a prognostic marker for non-small cell lung cancer9 and pancreatic cancer10 patients treated with a gemcitabine-based chemotherapy. CMPK1 has been observed in different subcellular locations of HeLa S3 cancer cells, as it predominantly localizes in the cytoplasm but can also enter the nucleus6. Subcellular localization of this protein may be important for cancer progression, but no study has yet revealed whether this has prognostic or predictive value in breast cancer. Therefore, further investigation may help to understand the clinical value of this marker, including its subcellular localization. In this study, we evaluated whether CMPK1 expression in different subcellular compartments is associated to patient prognosis in TNBC patients by IHC analysis of TMA derived from different academic medical centers in Europe. In order to elucidate the molecular pathways associated to subcellular localization of CMPK1, we performed GSEA correlation analysis on a subset of tumors incorporated in the TMA, of which gene expression data was available. A schematic representation of this workflow is shown in Fig. 1.
Results
Expression and subcellular localization of CMPK1 in triple negative breast cancer tissues.
Analysis of the staining results of an anti-CMPK1 antibody on breast tissues showed that CMPK1 prevalently stained epithelial cells of the mammary gland (normal cells: Fig. S1; carcinoma cells: Fig. S2A–D). When analyzing breast carcinoma tissues, both nuclear and cytoplasmic localization of CMPK1 protein was observed, as previously described in cancer cell lines6. Cytoplasmic CMPK1 (cCMPK1) was generally ubiquitous, as the majority of tissues (80.2%) expressed cCMPK1 in more (>) than 70% of carcinoma. Regarding intensity distribution, only a small percentage (9.5%) of tissues was found negative or showed a faint staining (7.0%), while most tissues displayed either weak (33.3%) or moderate (35.5%) cCMPK1 staining. Only 14.7% of cores showed strong cCMPK1 staining intensity (Fig. S2E–F). On the other hand, nuclear CMPK1 (nCMPK1) staining showed differences in terms of quantity of stained carcinoma cells and staining intensity. Nearly a third (29.7%) of cores did not show any nCMPK1 staining, while positive tissues displayed large heterogeneity in the number of stained tumor cells: 10.6% of tissues showed staining in 1–10% of tumor cells, while 12.8% and 17.2% displayed a number of stained carcinoma cells between 11–25% and 26–50%, respectively. One third (i.e. 29.7%) of tissue cores showed nCMPK1 staining in more than 50% of carcinoma cells (8.8% and 20.9% of the tumors showed expression in 51–70% and >70% of the tumor cells, respectively; Fig. S2E). When assessing nCMPK1 staining intensity distribution, the majority of positive tissues showed weak (16.5%), moderate (23.4%) and strong (24.5%) stainings, with only a small percentage (5.9%) showing faint staining (Fig. S2F). Overall these data show that, while cCMPK1 presents a generally ubiquitous and homogeneous staining, nCMPK1 staining varies widely in terms of stained tumor cells and staining intensity.
Differences between the TMA datasets. We next compared the three TMA datasets with respect to possible differences in clinical and histopathological parameters (Table 1). The RUMC and CUH cohorts comprised Scientific Reports | 6:32027 | DOI: 10.1038/srep32027
2
www.nature.com/scientificreports/ Total n of patients
RUMC*
CUH*
EMC*
Total*
48 (100.0)
83 (100.0)
267 (100.0)
398 (100.0)
Age (years) ≤40
7 (14.6)
12 (14.5)
65 (24.3)
84 (21.1)
41–55
18 (37.5)
22 (26.5)
113 (42.3)
153 (38.4)
56–70
19 (39.6)
29 (34.9)
67 (25.1)
115 (28.9)
4 (8.3)
20 (24.1)
22 (8.3)
46 (11.6)
>70 Menopausal status Premenopausal
18 (37.5)
24 (28.9)
155 (58.0)
197 (49.5)
Postmenopausal
30 (62.5)
59 (71.1)
112 (42.0)
201 (50.5)
Tumor size pT1
22 (45.8)
49 (59.1)
107 (40.1)
178 (44.7)
pT2 + pTx
24 (50.0)
30 (36.1)
147 (55.0)
201 (50.5)
pT3 + pT4
2 (4.2)
4 (4.8)
13 (4.9)
19 (4.8)
Good
0 (0.0)
2 (2.4)
4 (1.5)
6 (1.5)
Moderate
5 (10.4)
8 (9.6)
44 (16.5)
57 (14.3)
Poor
32 (66.7)
54 (65.1)
213 (79.8)
299 (75.1)
Unknown
11 (22.9)
19 (22.9)
6 (2.2)
36 (9.1)
Tumor Grade
Lymph node positivity 46 (95.8)
83 (100.0)
202 (75.6)
331 (83.2)
1 to 3
0
2 (4.2)
0 (0.0)
44 (16.5)
46 (11.6)
>3
0 (0.0)
0 (0.0)
21 (7.9)
21 (5.2)
Adjuvant chemotherapy Yes
2 (4.2)
40 (48.2)
83 (31.1)
125 (31.4)
No
46 (95.8)
43 (51.8)
184 (68.9)
273 (68.6)
Table 1. Clinical and histopathological characteristics of patients included in the TMAs. *Data are displayed as number (percentage). Acronyms: CUH: Copenhagen University Hospital; EMC: Erasmus University Medical Center; RUMC: Radboud University Medical Center.
a higher number of postmenopausal women (χ2 test P
