Quantitative Targeted Proteomics of Pancreatic Cancer: Deoxycytidine

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Quantitative Targeted Proteomics of Pancreatic Cancer: Deoxycytidine kinase protein level correlates to progression free survival of patients receiving gemcitabin treatment Ken Ohmine, Kei Kawaguchi, Sumio Ohtsuki, Fuyuhiko Motoi, Hideo Ohtsuka, Junichi Kamiie, Takaaki Abe, Michiaki Unno, and Tetsuya Terasaki Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00282 • Publication Date (Web): 17 Aug 2015 Downloaded from http://pubs.acs.org on August 20, 2015

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Molecular Pharmaceutics

Quantitative Targeted Proteomics of Pancreatic Cancer: Deoxycytidine kinase protein level correlates to progression-free survival of patients receiving gemcitabine treatment Ken Ohmine†∫, Kei Kawaguchi‡∫, Sumio Ohtsuki§, Fuyuhiko Motoi‡, Hideo Ohtsuka‡, Junichi Kamiie¶, Takaaki Abe£, Michiaki Unno‡ and Tetsuya Terasaki†*

† Membrane Transport and Drug Targeting Laboratory, Tohoku University Graduate School of Pharmaceutical Sciences, Sendai, Japan ‡ Division of Hepato-Biliary-Pancreatic Surgery, Tohoku University Graduate School of Medicine, Sendai, Japan § Department of Pharmaceutical Microbiology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan ¶ Laboratory of Veterinary Pathology, Azabu University School of Veterinary Medicine, Sagamihara, Japan £ Department of Clinical Biology and Hormonal Regulation, Tohoku University Graduate School of Medicine, Sendai, Japan

ACS Paragon Plus Environment

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Abstract

The purpose of the present study is to identify the determinant(s) of gemcitabine (dFdC)sensitivity in pancreatic cancer tissues of patients treated with dFdC alone and in pancreatic cancer cell lines exposed to dFdC in vitro. Protein expression levels of 12 enzymes and 13 transporters potentially involved in transport and metabolism of dFdC in pancreatic cancer cell lines and tissues were quantified by means of our LC-MS/MS-based quantitative targeted proteomics technology. Protein expression levels of deoxycytidine kinase (dCK), uridine monophosphate-cytidine monophosphate (UMP-CMP) kinase, cytosolic nucleotidase III (cN-III) and equilibrative nucleoside transporter 1 (ENT1) were significantly correlated with IC50 or 1/IC50 in 5 cell lines with different sensitivities to dFdC (p < 0.05). Expression levels of the selected proteins in pancreatic cancer tissues of 10 patients with different progression-free survival (PFS) (49 - 955 days) were quantified and their relationship with PFS was examined. Only the protein expression level of dCK was significantly correlated with PFS (p < 0.05). Multiple regression analysis was also performed, and combinations of ENT1, UMP-CMP kinase, CTPS1 and dCK were highly correlated with PFS. Our results indicate that the protein expression level of dCK in pancreatic cancer tissue is a good predictor of PFS, and thus dCK may be the best biomarker of dFdC sensitivity in pancreatic cancer patients treated with dFdC, although other proteins would also contribute to dFdC-sensitivity at the cellular level in vivo and in vitro.

Keywords gemcitabine; drug resistance; pancreatic cancer; targeted proteomics; deoxycytidine kinase


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Introduction Pancreatic cancer is highly malignant, and patients have a short survival period and a low 5year survival rate.1,

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The pyrimidine analogue gemcitabine (2’,2’-difluoro-2’-doxycytidine,

dFdC) has become a standard chemotherapeutic agent for pancreatic cancer, though it provides only a small benefit in terms of the 5-year survival rate, which is increased from 1% to 4%.2 Various combination chemotherapies, including dFdC, have not improved the situation. In contrast, TS-1, which consists of 5-FU and a specific inhibitor of its inactivating enzyme, improved the pharmacokinetics of 5-FU and achieved a chemotherapeutic outcome comparable to that of dFdC alone3-5. Therefore, understanding the mechanism(s) of dFdC sensitivity in pancreatic cancer may enable us to develop combination therapy that will improve the chemotherapeutic outcome in pancreatic cancer patients, compared with dFdC alone. dFdC is incorporated into cancer cells, where it is converted into the active triphosphate form (dFdCTP) by sequential phosphorylation, and dFdCTP exerts cytotoxicity through inhibition of DNA synthesis by interfering with the incorporation of endogenous dCTP into DNA.6,

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Therefore, we can envisage five possible approaches to overcome the dFdC sensitivity of cancer cells: 1) increase in incorporation of intact dFdC into cancer cells, 2) increase in phosphorylation of intracellular dFdC, 3) decrease in inactivation of intracellular intact or phosphorylated dFdC, 4) decrease in efflux of intracellular dFdC or its metabolites, and 5) decrease in synthesis of intracellular dCTP. As shown in Fig. 1, various proteins would be involved in each case. It is known that decrease in protein expression level or inactivation of deoxycytidine kinase (dCK), which converts dFdC to dFdC monophosphate, is the major cause of acquired dFdC resistance in several pancreatic cancer cell lines exposed to dFdC.8,

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Furthermore, high levels of

immunohistochemically detected dCK were significantly associated with longer survival among


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patients who received dFdC.10 Equilibrative nucleoside transporters (ENTs) and ribonucleotide reductase (RR) were also reported to be involved in resistance in other pancreatic cancer cell lines.11, 12 ENT1 transports nucleosides and several nucleoside analogs including dFdC into cells. Indeed, higher expression of ENT1 was found to increase the sensitivity of cells to dFdC,13 and reduced expression of ENT1 gene was reported to correlate with shorter survival of pancreatic cancer patients.14 Nevertheless, it was reported that resistant pancreatic cell lines expressed higher levels of ENT1 mRNA than sensitive cell lines, and suppression of ENT1 mRNA did not affect dFdC sensitivity in a pancreatic cancer cell line.12, 15, 16 Higher expression of RRM1 or M2 was found in several resistant pancreatic cancer cell lines and in pancreatic cancer patients with shorter survival, but the immunohistochemically determined level of RRM1 showed no correlation with survival among patients receiving dFdC.10,

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These complicated and

contradictory results can be attributed not only to differences between cancer tissues and cultured cells, and between protein and mRNA levels, but also to involvement of multiple proteins in activation and inactivation processes of dFdC. Given this complexity, a reasonable strategy to identify the key processes and proteins that determine differences in dFdC chemosensitivity would be comprehensive protein expression analysis by means of quantitative proteomics. Takadate et al. identified Nm23/nucleoside diphosphate kinase-A as a dFdC-sensitivity marker by means of comprehensive proteome analysis in dissected pancreatic cancer tissues.17 However, other molecules included in Fig. 1 were not found in that study.17 This suggests that general quantitative proteomics technology is not sufficiently sensitive to analyze all the metabolizing enzymes and transporters involved in dFdC activation and inactivation.


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An LC-MS/MS-based absolute quantitation method for proteins, so-called quantitative targeted absolute proteomics (QTAP), has recently been developed, and is suitable to determine the expression levels of multiple proteins in several tissues or cell lines.18, 19 In this method, the number of molecules that can be analyzed is limited compared with general quantitative proteomics, but the sensitivity is considerably higher. Furthermore, this method can determine absolute amounts of proteins with greater accuracy. We previously used this LC-MS/MS-based protein quantification method to determine the concentrations (fmol/µg protein) of multiple major proteins that are expressed in pancreatic cancer cells.8 Therefore, we anticipated that regression analysis of the accurately determined absolute amounts of the proteins shown in Fig. 1 with IC50 and/or survival would enable us to identify the key determinants of dFdC-sensitivity. As well as the method of quantitation, selection criteria for tissue samples would also be important. We previously demonstrated that dCK contributes to acquisition of dFdC resistance in pancreatic cancer cells8, and in this study, we focus on the relation between the clinical course and the protein expression level of membrane transporters and dFdC-related enzymes, including dCK, in pancreatic cancer tissues. However, it must be recognized that multiple factors influence clinical outcomes. For example, it was reported that 5-FU inhibited the de novo pathway and increased dFdC uptake activity with activation of the salvage pathway20 (Fig. 1). Here, in order to examine the relation between the clinical course and expression levels of various molecules in pancreatic cancer tissues, we retrospectively selected only cases that were chemo-naive before dFdC administration. Thus, the purpose of this study is to determine the expression levels of proteins involved in the activation, inactivation and transport of dFdC in pancreatic cancer cell lines and pancreatic cancer tissues, and to compare the results with IC50 of cultured cells or survival of cancer


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patients in order to identify the critical process(es) and protein(s) that regulate dFdC sensitivity in cells and tissues.

Experimental Section Reagents All peptides used for quantification of enzyme and transporter proteins were purchased from Thermo Fisher Scientific (Sedantrabe, Germany). All other reagents were commercial products of analytical grade.

Sample preparation of pancreatic cancer cell lines Five human pancreatic adenocarcinoma cell lines, PK9, CFPac-1, PK1, SUIT-2 and AsPC-1 (Table 1), were seeded onto non-coated tissue culture dishes (BD Biosciences Bedford, MA, USA) at the concentration of 1.5 x 104 cells/cm2, and cultured in RPMI1640 (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Moregate, Bulimba, Australia) at 37 ºC in a humidified atmosphere of 5% CO2 in air. The culture medium was exchanged every two days. Cells in log growth phase were centrifuged and washed with phosphate-buffered saline (PBS). The pellet was suspended in 50 mM Tris-HCl buffer (pH 7.4), containing 500 µM phenylmethylsulfonyl fluoride (PMSF) and 2 mM dithiothreitol (DTT). The cells were placed in a chamber (Central Scientific Commerce, Tokyo, Japan) under a nitrogen pressure of 800 p.s.i., for 15 min at 4 ºC and cell lysis was performed by rapid decompression. The lysed cells were centrifuged at 10,000 g for 10 min at 4 ºC and the supernatant was


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collected. After centrifugation at 100,000 g for 60 min at 4 ºC, the cell supernatant (cytosolic fraction) and precipitate (membrane fraction) were used for quantification of enzyme and transporter proteins.

Sample preparation of pancreatic cancer tissues To evaluate the effect of dFdC in vivo, 10 pancreatic cancer patients were arbitrarily selected from 58 surgical patients in Tohoku University Hospital, Japan from 2004 to 2006. Patients’ characteristics are summarized in Table 2. PFS (day) was defined as the period from the date of surgery to detection of obvious recurrence. Inclusion criteria were as follows: (1) histologically diagnosed as pancreatic ductal adenocarcinoma; (2) no neo-adjuvant therapy before operation; (3) no prior treatment before dFdC as adjuvant therapy; (4) sufficient sample size for LC-MS/MS (at least 0.05 g). Patients basically received dFdC 800 – 1000 mg/m2 on days 1, 8 and 15 every 28 days. The administration schedule was adjusted as necessary according to the patients’ general condition. Before administration of dFdC, patients underwent physical examinations and laboratory testing. Toxicity of dFdC in patients was evaluated according to the Common Toxicity Criteria (CTCAE, version 4.0). dFdC treatment was continued until disease progression was observed, unacceptable toxicity levels were reached, consent was withdrawn, or in the event of a decision to do so by the physicians. The research protocols for the present study were approved by the Ethics Committees of Tohoku University School of Medicine and the Graduate School of Pharmaceutical Sciences, Tohoku University. The study has been carried out in accordance with the Declaration of Helsinki.


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Frozen pancreatic cancer tissue was dissected and homogenized using a Potter-Elvehjem homogenizer in buffer consisting of 10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 1.5 mM MgCl2, 1 mM PMSF, and a protease inhibitor cocktail (Sigma Chemical Co., St. Louis, MO). The homogenized tissue was centrifuged at 10,000 g for 10 min at 4 ºC and the supernatant was collected. After centrifugation at 100,000 g for 60 min at 4 ºC, the cell supernatant (cytosolic fraction) and precipitate (membrane fraction) were used for quantification of enzyme and transporter proteins. Protein concentrations were measured by the Lowry method using the DC protein assay reagent (Bio-Rad, Hercules, CA).

Quantification of proteins by LC-MS/MS Prepared cytosolic and membrane fractions were alkylated and digested with trypsin according to the reported method.18 The absolute amounts of enzymes were determined by means of the multiplexed MRM method as described previously.8 The absolute amounts of transporters listed in Table S1 were determined by means of the multiplexed MRMHR method. The amino acid sequences and m/z values of the precursor ion and four product ions for each targeted protein are given in Table S1. The tryptic digests were acidified with formic acid and isotope-labeled peptides (10 fmol) were spiked into 1 µg of the digest from membrane fraction to provide internal references, then the digests were analyzed by LC-MS/MS. Samples for the calibration curve were prepared by serial dilution of synthetic peptides (1, 5, 10, 50, 100, 500, and 1000 fmol) spiked into 100 fmol of an internal standard peptide for enzymes, and serial dilution of synthetic peptides (0.1, 0.5, 1, 5, 10, 50, and 100 fmol) spiked into 10 fmol of an internal standard peptide for transporters. The sample analysis for transporters was automated by


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coupling a Q-ToF mass spectrometer (TripleToFTM5600, AB Sciex) to an Eksigent nanoLCUltra system. Each sample was injected onto a reversed-phase HPLC column (75 µm x 15 cm ChromXP X18-CL 3 µm, Eksigent); for conditions, see Table S2. Multiple products derived from single peptides were monitored at specific m/z transitions (Tables S1). Individual signal peaks were identified on the basis of equal retention times in each transition of multiple product ions.

Cytotoxicity assay The growth-inhibitory effect of dFdC on each cell line was assessed by means of colorimetric assay using a Cell Counting Kit (Dojindo, Kumamoto, Japan). Briefly, the cells were seeded on 96-well plates (1.6 x 104 cells/cm2) in culture medium containing 10% FBS. After 24 hr, the cells were incubated with 100 µL of culture medium containing dFdC (0, 0.3, 1, 3, 10, 30, 100, 300, 1000 and 3000 nM) for 72 hr. After incubation with dFdC, 10 µL of WST-8 regent was added, and incubation was continued for 4 hr. Cell viability was determined according to the manufacturer’s instructions. Absorbance was measured at 450 nm with a microplate reader (Model 680, Bio-Rad Laboratories, Hercules, CA, USA). Inhibition rate was plotted against dFdC concentration and the IC50 value was calculated from inhibition rate of just above (A) and below (B) 50% and concentration at A (a) and B (b) based on the following formula. IC50 (nM) = 10 ^ {Log (a/b) x (0.5 - B) / (A - B) + Log (b)}

Data analysis


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The ion counts in the chromatograms were determined using the quantitation procedures in the Analyst software package version 1.5 and MultiQuant (AB Sciex). Quantification values of each target peptide were determined with a set of MRM transitions by calculating the ratios of peak areas to those of isotope-labeled peptides as described previously.18 Then, the amount of each protein was determined as an average of three or four MRM transitions. All data are presented as the mean ± SEM. Regression analysis of protein expression levels and IC50 or PFS was performed by JMP software, version 11. Single regression analysis was performed for dCK and multiple regression analysis was performed for combinations of dCK with two other proteins among UMP-CMP kinase, ENT1, cN-III and CTPS1, which were expressed in all 5 cell lines and in 8 tissues (excluding two tissue samples in which the amounts of tryptic digest from membrane fraction were insufficient and ENT1 was not quantified).

Results Concentrations of selected proteins in pancreatic cancer cell lines and their correlation to in vitro dFdC chemosensitivity index To evaluate the correlation between IC50 as a dFdC chemosensitivity index and the absolute concentrations of proteins potentially influencing sensitivity to dFdC, 5 immortalized human pancreatic cancer cell lines covering an 11.8-fold range of IC50 values (Table 1) were selected. The absolute protein concentrations of 12 enzymes in cytosolic fraction and 13 transporters in membrane fraction of 4 human pancreatic cancer cell lines were determined, and those for PK9


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cells were taken from our previous report.8 The results for all cell lines are summarized in Tables S3 and S4. In the cytosolic fraction, 8 enzymes in the salvage pathway, which are directly involved in metabolism of dFdC, and 4 enzymes in the de novo pathway, which are involved in dFdCsensitivity by affecting intracellular dCTP level, were quantified. The protein concentrations of 5 salvage enzymes, i.e., dCK, uridine monophosphate-cytidine monophosphate (UMP-CMP) kinase, deoxycytidylate deaminase (DCTD), cytosolic nucleotidase II (cN-II), and cN-III, and those of 3 de novo enzymes, i.e., RRM1, cytidine triphosphate synthetase 1 (CTPS1) and CTPS2, were evaluable in all cell lines. Expression of cytidine deaminase (CDA) cN-IA, cN-IB and RRM2 was not detected in any of the cell lines. Protein concentrations of the 8 cytosolic enzymes that were detected in all cell lines were compared with IC50 or 1/IC50 values of dFdC to identify proteins potentially contributing to dFdC sensitivity (Figs. 2 and 3). A positive correlation with IC50 is expected in the case of proteins for which an increase in concentration favors chemoresistance, while a positive correlation with 1/IC50 is expected in the case of proteins for which an increase in concentration favors chemosensitivity. As shown in Fig. 3, the protein concentrations of the kinases dCK and UMP-CMP kinase were significantly positively correlated with 1/IC50, but not with IC50, suggesting that increased protein concentrations of these kinases promote dFdC chemosensitivity by activating dFdC via phosphorylation. On the other hand, the protein concentration of cN-III, which is involved in dephosphorylation of dFdC monophosphate, was significantly negatively correlated with 1/IC50, which suggests that increase of cN-III contributes to decreased dFdC chemosensitivity by inactivating dFdC. Protein concentrations of the other proteins in this group were not significantly correlated with either IC50 or 1/IC50.


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In the membrane fraction, we quantified the protein concentrations of 5 nucleoside transporters and 8 ATP-binding cassette transporters, which are expected to transport dFdC and/or its metabolites. Among them, only ENT1, which transports dFdC, was detected in all cell lines. The protein concentration of ENT1 was significantly positively correlated with IC50, but not with 1/IC50, suggesting that an increase in protein concentration of ENT1 decreases dFdC chemosensitivity. Multidrug resistance-associated protein 1 (MRP1), MRP2, MRP3, MRP4 and breast cancer resistance protein (BCRP) were detected in one to four cell lines, but the protein expression levels of the other transporters were under the detection limit in all cell lines.

Concentrations of selected proteins in pancreatic cancer tissues and their correlation to progression-free survival of patients treated with dFdC To clarify the in vivo expression profile of proteins potentially involved in dFdC sensitivity, the absolute protein concentrations of 12 enzymes in cytosolic fraction and 13 transporters in membrane fraction of pancreatic cancer tissues from the 10 pancreatic cancer patients were determined (Tables S5 and S6). Among enzymes in the salvage pathway, 6 proteins (dCK, UMP-CMP kinase, CDA, DCTD, cN-II and cN-III) were detected in cytosolic fraction of at least 8 samples. Among enzymes in the de novo pathway, CTPS1 and CTPS2 were detected in cytosolic fraction of at least 8 samples. Therefore, to identify proteins contributing to dFdC chemosensitivity in vivo, the quantitative values of these 8 enzymes were compared with PFS (Fig.4). Among them, the protein concentration of dCK was correlated with PFS (R2 = 0.699, p = 0.0026). RRM1 and M2


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were under the detection limit in all samples, although they had been detected in pancreatic cancer cell lines. cN-IA and IB were under the detection limit in all samples and cell lines. In the case of membrane fraction, only 8 samples were available for quantification, because the amounts of tryptic digests were insufficient for the other 2 samples (No.1 and 8). Among the transporters examined, only ENT1 was detected in all 8 samples. However, the protein concentration of ENT1 did not significantly correlate with PFS. MRP6 was detected in two samples from patients with short survival, and the other 11 transporters were under the detection limit in all samples.

Correlation of the expression levels of combinations of proteins with dFdC sensitivity Since the salvage and de novo pathways involve sequential reaction with multiple proteins, we conducted multiple regression analysis of dCK with any two other proteins among UMP-CMP kinase, ENT1, cN-III and CTPS1, whose expression levels were significantly correlated with IC50, 1/IC50 or PFS, to search for additive effects. The combinations for which the coefficient of determination, R2, was greater than that of dCK alone are shown in Tables 3 and 4. For pancreatic cancer cell lines, the combination of UMP-CMP kinase, ENT1 and dCK gave the best correlation with 1/IC50, but the improvement over dCK alone was small (Table 3). In pancreatic cancer tissues, the combination of ENT1, CTPS1 and dCK gave the best correlation with PFS (R2 = 0.965, p = 0.0023) (Table 4).

Discussion


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In this study, we found that the absolute protein concentrations of dCK in pancreatic cancer cell lines and tissues were highly correlated with IC50 and PFS, respectively, which were taken as indexes of dFdC sensitivity. Although multiple processes and proteins may influence dFdC sensitivity (Fig. 1), this result suggests that phosphorylation of dFdC by dCK may be a key determinant of dFdC sensitivity both in vitro and in vivo. The absolute concentrations of several other proteins, such as UMP-CMP kinase and cN-III (involved in dFdC phosphorylation) and ENT1 (involved in transport of nucleoside analogs including dFdC) were also significantly correlated with dFdC-sensitivity index in vitro or in vivo in this study, but with lower coefficients of determination than that of dCK. These results suggest that dCK may be the best marker for dFdC sensitivity of pancreatic cancer samples among the molecules examined, and it is therefore a promising candidate target to increase the dFdC sensitivity of pancreatic cancer. The phosphorylation process in the salvage pathway, which converts dFdC to dFdCTP, is expected to be one of the main pathways determining dFdC-sensitivity, and 2 kinases (dCK and UMP-CMP kinase) and 1 phosphatase family (cNs) are involved in this process (Fig. 1).21-23 We found that the protein concentration of dCK was highly correlated with both in vitro and in vivo indexes of dFdC sensitivity (Figs. 2, 3 and 4). This suggests that the phosphorylation of dFdC by dCK is the main determinant of dFdC sensitivity in both cultured cells and cancer tissue, and therefore the protein concentration of dCK in cancer tissue should be a good marker for predicting the dFdC sensitivity of pancreatic cancer patients. This is consistent with the previous finding that high levels of immunohistochemically detected dCK in cancer tissues were significantly associated with longer survival among patients who received dFdC.10 Unlike dCK, the protein concentrations of UMP-CMP kinase and cN-III showed significant correlations with dFdC sensitivity only in cell lines. This indicates that the contribution of phosphorylation


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processes to dFdC sensitivity is different in vitro and in vivo. In the case of the cell lines (Figs 2 and 3), these three proteins cooperatively influence dFdC sensitivity, whereas in cancer tissues, the protein concentration of dCK appears to be the main determinant. Nevertheless, in multiple regression analysis, inclusion of UMP-CMP kinase with dCK only modestly increases the correlation coefficient both in vivo and in vitro (Tables 3 and 4). This suggests that, even in cancer tissues, phosphorylation of dFdCMP by UMP-CMP kinase may play a minor role in determining dFdC sensitivity, though the role of dCK is predominant. Protein concentration of ENT1 showed a positive correlation only with IC50 in vitro, but not with PFS. ENT1 is a major nucleoside transporter, and also transports several nucleoside analogs including dFdC.24, 25 Increased ENT1 levels in cell lines and tissues cause an increase of dFdC sensitivity through enhancement of dFdC incorporation into the cells. 10, 11, 13, 26 However, it was also reported that expression levels of ENT1 positively correlated with IC50 of dFdC, in agreement with the finding in the present study. 15 It is known that ENT1 transports precursors of dCTP such as uridine, cytidine and deoxycytidine into cells, and intracellular dCTP levels influence the cytotoxicity of Ara-C, which has almost the same action mechanism as dFdC.27 Nakano et al. reported that mRNA levels of ENT1, RRM1 and M2 increased concomitantly with a decrease in mRNA level of dCK, which serves as a salvage enzyme. novo enzymes mainly involved in dCTP synthesis.

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RRM1 and M2 are de

Therefore, ENT1 might mainly regulate

intracellular dCTP level, but not intracellular dFdC level, in conjunction with RRM1 and M2 as de novo enzymes, and so it might indirectly affect the dFdC sensitivity of cell lines in vitro. Our findings show that RRM1 and M2 protein levels in pancreatic cancer tissues are lower than those in pancreatic cancer cell lines. Protein concentrations of RRM1 were less than 0.2 fmol/µg protein in all tissue samples, although they were more than 0.2 fmol/µg protein in all


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cell lines (Tables S3 and S5). Protein concentrations of RRM2 were also less than 0.2 fmol/µg protein in all tissue samples, although they were more than 0.7 fmol/µg protein in 3 cell lines (Tables S3 and S5). Pancreatic cancer is well known as an ischemic cancer and its microenvironment is hypoxic.

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It was also reported that RR activity and intracellular dCTP

decrease under hypoxic conditions.30, 31 Therefore, our findings in this study (Figs 2 and 4) that the protein concentration of ENT1 showed a significant correlation with in vitro IC50 values, but not with in vivo survival, might be explained by the possibility that ENT1 was unable to influence dFdC sensitivity through dCTP synthesis in cancer tissues due to the hypoxia-induced down-regulation of RR. As shown in Fig. 4, the apparent relationship of protein concentration of CTPS1 with PFS was not statistically significant. CTPS1 generally controls the levels of both intracellular CTP (an RNA precursor) through metabolism of UTP to CTP and intracellular dCTP (a DNA precursor) through subsequent reduction of ribonucleotide, mainly mediated by RR, as shown in Fig. 1.

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However, the present results suggest that CTPS1 in pancreatic cancer tissues may control only the CTP level, due to the low level or loss of RR under hypoxia, as mentioned above. Indeed, cyclopentenyl cytosine (CPEC), which is a CTPS inhibitor, did not enhance the efficacy of dFdC for tumor regression in a pancreatic cancer cell xenograft model. 33 On the other hand, the level of intracellular dCTP is mainly controlled by salvage enzymes, including dCK, not de novo enzymes, such as CTPS and RR. Thus, it seems possible that the intracellular dCTP level, controlled by dCK, affects expression of CTPS as a regulator of RNA synthesis, resulting in the observed correlation between CTPS concentration and survival in vivo. Correlation analysis cannot distinguish between a cause and a result of target drug efficacy. Further work will be necessary to examine possible interactions among CTPS, CTP, dCTP and dFdC.


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In this study, the protein expression level of dCK was highly correlated with PFS, and it may therefore be available as an in vivo marker for dFdC sensitivity. Other reports have indicated that mRNA expression ratios of combinations of molecules including dCK showed higher correlations with in vitro or in vivo dFdC-sensitivity markers compared with the mRNA expression level of any of the molecules alone.12, 34-36 However, protein levels of dCK showed a good correlation with dCK specific activities in several kinds of cells, whereas mRNA levels of dCK showed no correlation with dCK activities in other cell lines.37, 38 Thus, protein level of dCK may be preferable as an in vivo dFdC-sensitivity marker. Nevertheless, we cannot rule out the possibility that the expression levels of combinations of proteins might be a significantly better marker than dCK alone to predict survival, as several combinations including UMP-CMP kinase, ENT1, CTPS1 and dCK gave better correlations with PFS, though not with IC50, than did dCK alone in this study. It should also be considered that other factors might influence the dFdC sensitivity of pancreatic cancer. For example, the severity of disease may have differed among patients, and the percentage of cancer vs. normal tissues may have varied markedly in the surgical materials used for the current study; in addition, dFdC exposure levels in patients and co-medications that interact with dFdC may also be important factors. However, the present study included only 10 patients with defined PFS. We estimate that PFS of pancreatic cancer patients treated with dFdC after resection is 12 months. This estimation is based on a previous study39 and our clinical experience. It is supposed that the patients with more than 12 months of PFS are dFdC effective group and less than 12 months are ineffective group. With a probability of an α error of 0.05, a standard deviation of 0.3 and a power of 0.80, At least 22 patients are required in each group. Consideration for a 10% loss of follow-up, we calculate that more than 50 patients with appropriate background information are required to increase statistical power to


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perform multiple regression analysis including these factors. Thus, further studies with a larger number of tissue samples will be needed to examine in detail the contributions of multiple proteins and other factors. In conclusion, we have determined the concentrations of multiple enzyme and transporter proteins potentially associated with dFdC sensitivity in pancreatic cancer cell lines and tissues by means of QTAP, and examined the correlation of these levels to in vitro and in vivo dFdCsensitivity indices. We identified two important determinant molecules that differently affect the sensitivity. One of them, ENT1, appeared to influence dFdC sensitivity only in cell lines in vitro, but not in tissues. The reason for this may be that nucleoside uptake process by ENT1 is limiting for intracellular dCTP level in vitro, but not in vivo, because RR activity is higher in vitro than in hypoxic cancer tissues. dCK was a determinant of dFdC sensitivity both in vitro and in vivo. Therefore, we suggest that dCK, rather than ENT1, is not only a potent marker for predicting dFdC sensitivity of pancreatic cancer tissues, but also the best candidate target for combination therapy. Drugs that induce dCK expression and/or activity, which could be identified by in vitro screening, might enhance the efficacy of dFdC in treatment of pancreatic cancer.


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Figure 1. Schematic illustration of transport and metabolism of dFdC and intrinsic nucleosides. The indicated transporter and enzyme proteins were quantified by LC-MS/MS. As ATP binding cassette transporters (ABC), multidrug resistance protein 1 (MDR1), multidrug resistanceassociated proteins (MRP) and breast cancer resistance protein (BCRP) were quantified. dFdCMP: dFdC monophosphate; dFdCDP: dFdC diphosphate; dFdCTP: dFdC triphosphate; dFdU: 2’,2’-difluorodeoxyuridine; dFdUMP: dFdU monophosphate; ENT: equilibrative nucleoside transporter; CNT: concentrative nucleoside transporter; CDA: cytidine deaminase; dCK: deoxycytidine kinase; cN, cytosolic 5’-nucleotidase; DCTD, deoxycytidylate deaminase; RR, ribonucleotide reductase; CTPS, CTP synthetase.


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Figure 2. Correlation between concentrations of selected proteins in pancreatic cancer cell lines and IC50 of dFdC. Coefficient of determination (R2) and p values for correlation of A) ENT1, B) dCK, C) UMP-CMP kinase, D) DCTD, E) cN-II, F) cN-III, G) RRM1, H) CTPS1 and I) CTPS2 were calculated from protein concentrations and IC50 values in 5 cell lines.


20

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3. Correlation between concentrations of selected proteins in pancreatic cancer cell lines and 1/IC50 of dFdC. Coefficient of determination (R2) and p values for correlation of A) ENT1, B) dCK, C) UMP-CMP kinase, D) DCTD, E) cN-II, F) cN-III, G) RRM1, H) CTPS1 and I) CTPS2 were calculated from protein concentrations and 1/IC50 in 5 cell lines.


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Figure 4. Correlation between concentrations of selected proteins in pancreatic cancer tissues and progression-free survival (PFS) of pancreatic cancer patients treated with dFdC. Coefficient of determination (R2) and p values of B) dCK, C) UMP-CMP kinase, D) CDA, E) DCTD, F) cNII, G) cN-III, H) CTPS1 and I) CTPS2 were calculated from protein concentrations and PFS (10


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samples). Protein concentrations of CDA and CTPS2 were under the detection limit in 2 samples and the concentrations were taken as equal to the detection limit for calculation of R2 and p values. R2 and p value of A) ENT1 were calculated from protein concentrations and PFS in 8 samples, since protein concentrations of 2 samples could not be evaluated.


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Table 1 Values of IC50 of dFdC in pancreatic cancer cell lines Cell lines

IC50 (nM)

Resistance ratio

PK9

3.53 ± 0.82

1

CFPac-1

4.92 ± 0.28

1.39

PK1

18.3 ± 2.2

5.18

SUIT-2

23.2 ± 7.8

6.57

AsPC-1

41.7 ± 9.8

11.8

IC50 in each cell line is the average ± S.E.M. (n=8). Resistance ratio means ratio of IC50 to that of PK9.


24

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 2 Patient information

Patient Age Gender No.

Dosage

PFS (day)

mg/m2

mg

Body surface Frequency area (m2)

1

66

M

49

1,000

1600

-

18

2

56

M

51

1,000

1500

1.463

7

3

66

F

94

1,000

1400

1.348

41

4

68

M

56

1,000

1600

1.577

13

5

69

M

154

1,000

1500

1.469

24

6

67

F

197

1,000

1200

1.254

22

7

57

F

406

1,000

1400

1.419

31

8

53

F

257

1,000

1400

1.374

25

9

85

M

287

1,000

1585

1.585

6

10

74

F

955

800

1200

1.531

-

PFS means the period from the date of surgery to detection of obvious recurrence. Patients were given dFdC 800 – 1000 mg/m2 as shown above on the following schedules: days 1, 8 and 15 every 28 days (Patient No. 1 – 5 and 8), days 1 and 8 every 21 days (Patient No. 6), and every 2 weeks (Patient No. 9 and 10). In the case of No. 7, initial administration was every 28 days and this was later changed to every 2 weeks. No patients received dFdC before surgery; they were treated with dFdC only after surgery. No data were recorded for dosage (mg) and body surface area in No.1 and for frequency in No. 10.


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Table 3 Results of regression analysis of concentration or reciprocal concentration of selected protein combinations against 1/IC50 for pancreatic cancer cell lines Independent variables

R2

p

dCK, UMP-CMP kinase, ENT1

1.00

0.0003

dCK, UMP-CMP kinase, 1/ENT1

1.00

0.0223

dCK, 1/UMP-CMP kinase, 1/ENT1

1.00

0.0488

dCK, UMP-CMP kinase, 1/CTPS1

0.999

0.0451

dCK, 1/cN-III, 1/ENT1

0.999

0.0480

dCK, 1/UMP-CMP kinase, ENT1

0.997

00651

dCK

0.989

0.0005

Single regression analysis of dCK and multiple regression analysis of dCK with two other proteins among UMP-CMP kinase, ENT1, cN-III and CTPS1 against 1/IC50 were performed. Combinations showing a higher coefficient of determination (R2) with 1/IC50 than that of dCK alone are listed.


26

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Table 4 Results of regression analysis of concentration or reciprocal concentration of selected protein combinations against PFS for pancreatic cancer tissues Independent variables

R2

p

dCK, 1/CTPS1, ENT1

0.965

0.0023

dCK, 1/CTPS1, 1/ENT1

0.959

0.0031

dCK, UMP-CMP kinase, CTPS1

0.741

0.0339

dCK, 1/UMP-CMP kinase, cN-III

0.726

0.0399

dCK, 1/UMP-CMP kinase, 1/CTPS1

0.724

0.0409

dCK, UMP-CMP kinase, cN-III

0.706

0.0490

dCK, UMP-CMP kinase, 1/CTPS1

0.704

0.0500

dCK

0.699

0.0026

Single regression analysis of dCK and multiple regression analysis of dCK with two other proteins among UMP-CMP kinase, ENT1, cN-III and CTPS1 against progression-free survival (PFS) were performed. Combinations showing a higher coefficient of determination (R2) with PFS than that of dCK alone are listed.


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AUTHOR INFORMATION Corresponding Author *Tetsuya Terasaki. Address: Membrane Transport and Drug Targeting Laboratory, Tohoku University Graduate School of Pharmaceutical Sciences, 6-3 Aoba, Aramaki, Aoba-ku, Sendai 980-8578, Japan. E-mail: [email protected] Tel: +81 22 795 6831. Fax: +81 22 795 6886.

Author Contributions ∫ These authors contributed equally.

Acknowledgment This study was supported in part by a Grant-in-Aid for JSPS Fellows, a Global COE Program from the Japan Society for the Promotion of Science, and a Grant for Development of Creative Technology Seeds Supporting Program for Creating University Ventures from Japan Science and Technology Agency. This study was also supported in part by the Industrial Technology Research Grant Program from the New Energy and the Industrial Technology Development Organization of Japan, and the Funding Program for Next Generation World-Leading Researchers by the Cabinet Office, Government of Japan.


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Abbreviations PFS, progression-free survival; dFdC, Gemcitabine; dFdCMP, dFdC monophosphate; dFdCDP, dFdC diphosphate; dFdCTP, dFdC triphosphate; dFdU, 2’,2’-difluorodeoxyuridine; dFdUMP, dFdU monophosphate; ABCs, ATP binding cassette transporters; MDR1, multidrug resistance protein 1; MRP, multidrug resistance-associated protein; BCRP, breast cancer resistance protein; ENT, equilibrative nucleoside transporter; CNT, concentrative nucleoside transporter; CDA, cytidine deaminase; dCK, deoxycytidine kinase; cN, cytosolic 5’-nucleotidase; DCTD, deoxycytidilate deaminase; RR, ribonucleotide reductase; CTPS, CTP synthetase; QTAP, quantitative targeted absolute proteomics


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to messenger RNA expression in blood cells from untreated patients with B-cell chronic lymphocytic leukemia. Biochemical pharmacology 2006, 71, (6), 882-90. 39.

Oettle, H.; Neuhaus, P.; Hochhaus, A.; Hartmann, J. T.; Gellert, K.; Ridwelski, K.;

Niedergethmann, M.; Zülke, C.; Fahlke, J.; Aming, M. B.; Sinn, M.; Hinke, A.; Riess, H. Adjuvant chemotherapy with gemcitabine and long-term outcomes among patients with resected pancreatic cancer: the CONKO-001 randamized trial. The Journal of the American Medical Association 2013, 310, (14), 1473-81.


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Table of Contents Graphic and Synopsis

The purpose of the present study is to identify the determinant(s) of gemcitabine (dFdC) sensitivity in pancreatic cancer tissues of patients treated with dFdC alone and in pancreatic cancer cell lines exposed to dFdC in vitro. Protein expression levels of 12 enzymes and 13 transporters potentially involved in transport and metabolism of dFdC in pancreatic cancer cell lines and tissues were quantified by means of our LC-MS/MS-based quantitative targeted proteomics technology. Protein expression levels of deoxycytidine kinase (dCK), uridine monophosphate-cytidine monophosphate (UMP-CMP) kinase, cytosolic nucleotidase III (cN-III) and equilibrative nucleoside transporter 1 (ENT1) were significantly correlated with IC50 or 1/IC50 in 5 cell lines with different sensitivities to dFdC (p < 0.05). Expression levels of the selected proteins in pancreatic cancer tissues of 10 patients with different progression-free survival (PFS) (49 - 955 days) were quantified and their relationship with PFS was examined.


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Only the protein expression level of dCK was significantly correlated with PFS (p < 0.05). Multiple regression analysis was also performed, and combinations of ENT1, UMP-CMP kinase, CTPS1 and dCK were highly correlated with PFS. Our results indicate that the protein expression level of dCK in pancreatic cancer tissue is a good predictor of PFS, and thus dCK may be the best biomarker of dFdC sensitivity in pancreatic cancer patients treated with dFdC, although other proteins would also contribute to dFdC-sensitivity at the cellular level in vivo and in vitro.


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Quantitative Targeted Proteomics of Pancreatic Cancer: Deoxycytidine

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