Ann Surg Oncol DOI 10.1245/s10434-014-4150-3
ORIGINAL ARTICLE – COLORECTAL CANCER
Prokineticin 1 Protein Expression is a Useful New Prognostic Factor for Human Sporadic Colorectal Cancer Toshiyuki Nakazawa, MD, Takanori Goi, MD, PhD, Yasuo Hirono, MD, PhD, and Akio Yamaguchi, MD, PhD First Department of Surgery, University of Fukui, Fukui, Japan
ABSTRACT Background. Hematogenous metastasis, regarded as closely related to angiogenic growth factors, is associated with colorectal cancer prognosis. The angiogenic growth factor prokineticin 1 (PROK1) has been cloned from endocrine cells. However, its protein expression in human malignant tumors has not been studied. The current study established the anti-PROK1 monoclonal antibody (mAb) and examined the relationship between the expression of PROK1 protein and human colorectal cancer. Methods. The expression of PROK1 protein was assessed in 620 resected sporadic colorectal cancer tissue samples by immunohistochemical staining with in-house-developed human PROK1 mAb to investigate the relationship of PROK1 expression to clinicopathologic factors, recurrence, and survival rate and to evaluate its prognostic significance. Results. The expression of PROK1 protein was detected in 36 % (223/620) of human primary colorectal cancer lesions but no in the healthy mucosa adjacent to the colorectal cancer lesions. According to the clinicopathologic examinations, the frequency of positive PROK1 expression was significantly higher in cases with serosal invasion, lymphatic invasion, venous invasion, lymph node metastasis, liver metastasis, hematogenous metastasis, and higher stage disease. The recurrence rate and prognosis for patients with PROK1 expression–positive lesions were significantly worse. In the Cox proportional hazard model, PROK1 expression was an independent prognostic factor. Conclusions. The expression of PROK1 protein was identified for the first time as a new prognostic factor in colorectal cancer.
Ó Society of Surgical Oncology 2014 First Received: 14 May 2014 T. Goi, MD, PhD e-mail: [email protected]
To improve prognosis in colorectal cancer treatment, measures taken against hepatic and other hematogenous metastases are of the utmost importance.1 Such measures include identification of patients at risk for hematogenous metastasis so that hematogenous metastases can be detected early and treated promptly with intensive postoperative adjuvant therapy and follow-up observation. According to a recently proposed hypothesis on the mechanism of colorectal cancer metastasis, tumor cells first detach from the primary lesion. This detachment is followed by disintegration of the basement membrane, migration of the tumor cells into the interstitium, and vascular invasion.2 A number of studies have reported many genes that might be involved in each step of metastasis based on molecular biologic analyses.3–5 Angiogenic growth factors are known to be important, and several angiogenic growth factors are thought to be involved in the development of colorectal cancer.6–8 Prokineticin 1 (PROK1), the protein examined in the current study, was first reported as an angiogenic growth factor by Ferrara.9 Analysis of PROK1 mRNA expression in healthy tissues showed that it is expressed in limited tissue types including the adrenal gland, ovary, testis, and other endocrine tissues.9 Vascular endothelial growth is promoted by PROK1 after its induction under hypoxic conditions, but growth activity and chemotactic activity are observed only in endocrine cells.9 We recently reported that the introduction of PROK1 into a colorectal cancer cell line frequently induces angiogenesis and hematogenous metastasis10 and that cellular infiltration is promoted by the autocrine mechanism.11 Other reports on PROK1 and malignant tumors have shown that PROK1 is related to prostate cancer and neuroblastoma metastasis and that it increases the malignancy of pancreatic duct cancer, suggesting that PROK1 activity is important in malignant tumors.12–14 Nevertheless, no studies have investigated the expression of PROK1 in human colorectal cancer. We therefore performed a detailed analysis of
T. Nakazawa et al.
PROK1 expression in 620 primary colorectal cancer lesions to assess the relationship between PROK1 expression and clinicopathologic factors, recurrence rate, and prognosis. MATERIALS AND METHODS Construction of Plasmid pGEX-PROK1 and Affinity Purification of Bacterial Extracts The total RNA was extracted from the colon cancer cells using guanidinium-thiocyanate. Single-strand cDNA prepared from 3 lg of total RNA using Moloney murine leukemia virus reverse transcriptase (Gibco-BRL, Rockville, MD, USA) with an oligo (dT) primer-14 was used as the template for the polymerase chain reaction (PCR).15 The products were used for 30 cycles of PCR amplification using two primers: the 50 primer, PROK1-AX, GGATC CATGAGAGGTGCCACGCGAGTCTCAATC of the published human PROK1 sequence9 and the 30 primer, PROK1BX, GAATTCAAAATTGATGTTCTTCAAGTCCATGG AGCAGCGG. Next, 35 cycles of denaturation (94 °C, 1 min), annealing (52 °C, 1.5 min), and extension (72 °C, 2.5 min) were carried out in a thermal cycler (PTC-100, Programmable Thermal Controller; NJ Research Inc., USA). Then, 50 ll of the PCR products that showed the relevant bands in reverse transcriptase (RT)-PCR analysis were visualized in ethidium bromide-stained 1.2 % agarose gels, purified, and cloned into pGEX2T vector (Pharmacia, Uppsala, Sweden).16 The plasmid constructs were confirmed by DNA sequencing. Fusion proteins were induced by adding isopropyl-B-Dthiogalactopyranoside to bacterial culture. These fusion proteins were purified by glutathione Sepharose 4B (Pharmacia, Uppsala, Sweden) affinity column.16 Also, PROK1 proteins were purified using thrombin (Pharmacia, Uppsala, Sweden). Western Blot Analysis For Western blot analysis, 5 lg of purified protein was run on a 10 % sodium dodecyl sulfate (SDS)-polyacrylamide gel and transferred to polyvinylidene difluoride (PVDF) membrane. After the electrophoretic transfer, the membrane was blocked overnight at 4 °C and incubated with the primary antibody. The protein bands were incubated with anti-PROK1 monoclonal antibody (mAb) (established by our department). Density was visualized by enhanced chemiluminescence according to the manufacturer’s instructions (Amersham, Piscataway, NJ, USA). In Vitro Tube Formation Assay The samples (fresh medium containing recombinant PROK1 protein; Shenandoah Company, Warwick,
Philadelphia, USA) (10 ng/ml) or the culture fluid plus mAb or normal mouse immunoglobulin G (IgG; Santa Cruz Biotechnology, USA) were added to wells of an angiogenesis kit (Kurabo Co., Neyagawa, Japan). On day 5, cells were fixed in 70 % ethanol and stained with antiCD31 mAb (Tubule staining kit; Kurabo Co., Neyagawa, Japan). For the evaluation of capillary tube formation (the stained tube-like structures), each well was photographed, and the total tube length was analyzed by the MacSCOPE program (Mitani Company, Fukui, Osaka, Japan).15 The experiments were repeated four times. Patients and Samples Surgical specimens and adjacent normal colorectal tissues were obtained from surgical resections from 620 patients with sporadic primary colorectal cancer in the First Department of Surgery, University of Fukui, Japan between 1990 and 2007. The ages of the 620 patients ranged from 22 to 95 years. Cancerous tissues and corresponding normal tissues were obtained at surgery. According to the tumor-nodemetastasis (TMN) classification,17 117 patients had stage 1 cancer, 199 had stage 2 cancer, 223 had stage 3 cancer, and 81 had stage 4 cancer. Because histopathologic findings varied within the same tumors, the diagnosis was based on the dominant pattern evaluated by two pathologists. All samples were fixed in 10 % paraformaldehyde (pH 6.8) for 24 h and embedded in paraffin. The eligibility criteria for the study specified histopathologic findings confirming primary colorectal cancer, resection of colorectal cancer with extended (D2 or D3) lymph node dissection,18 histologic curative resection (stages 1–3), an Eastern Cooperative Oncology Group performance status (PS) of 0 or 1, no chemotherapy or radiotherapy before surgical resection, 5-fluorouracil-based chemotherapy administered after surgical resection to patients with stage 3 or 4 disease, and no chemotherapy administered to patients with stage 1 or 2 disease after surgical resection. All the patients were followed up for recurrence at regular intervals for 5 years and underwent chest X-ray, computed tomography, and colonoscopy. Immunohistochemical Study Paraffin sections 4 lm thick were deparaffinized with xylene and then dehydrated through a graded ethanol series. Endogenous peroxidase activity was blocked by incubation for 30 min with 1 % hydrogen peroxidase in methanol. These hydrate sections were incubated in a dilution of normal goat serum at room temperature for 20 min to reduce nonspecific staining and then incubated with anti-PROK1 mAb for 1 h. After washing with tris buffered saline, and analyzed for the expression of PROK1 protein by the
PROK1 as a New Prognostic Factor
ChemMate method described earlier, the slides finally were lightly counterstained with hematoxylin. The expression was interpreted as positive when the protein was expressed in more than 30 % of all cancer cells using Image J Software (http://rsb.info.nih.gov/ij/).
clinicopathologic factors showed that gender, age, histologic type, and peritoneal metastasis were not related to PROK1 expression. However, PROK1 expression was observed more frequently in patients with lymphatic invasion, venous invasion, tumor depth, lymph node metastasis, hematogenous metastasis, and TMN stage (Table 1).
Statistical Analysis The association of PROK1 expression with histologic type, primary tumor, serosal invasion, lymphatic invasion, venous invasion, lymph node metastasis, peritoneal metastasis, liver metastasis, and hematogenous metastasis was assessed by cross-tabulation, and statistical significance was determined by the v2 test or Student’s t test using Stat Mate IV (ATMS Co., Ltd., Tokyo, Japan). Life-table analysis was performed using the Kaplan–Meier technique. Life time was overall survival time. The outcomes from different groups of patients were compared by the log rank test using Stat Mate IV (ATMS Co., Ltd., Tokyo, Japan). The Cox proportional hazards model was used in multivariate regression analyses of the survival date using SPSS software (IBMM SPSS Statistics, IBM Corp., Armonk, NY, USA). Differences were considered significant at p values lower than 0.05. RESULTS Development of a Human Monoclonal Anti-PROK1 Antibody After removal of spleen cells from mice immunized with a PROK1-GST fusion protein, the spleen cells were fused with NS-1 myeloma cells. Subcloning and limiting dilution were repeated three times to generate an anti-PROK1 mAb. This antibody detected a protein in the lanes of a gel containing PROK1 and the PROK1-GST fusion (Fig. 1a). PROK1 Protein Expression in Human Colorectal Cancer Primary Lesions and Adjacent Healthy Mucosa The PROK1 protein was expressed in primary colorectal cancer lesions but not in the surrounding human healthy mucosa. A representative example is shown in Fig. 1b. We observed PROK1 protein expression in 223 of the 620 human colorectal cancer cases examined. PROK1 Protein Expression in Human Colorectal Cancer Tissue and the Associated Clinicopathologic Factors Examination of the relationship between PROK1 protein expression in primary colorectal cancer lesions and
Relationship Between PROK1 Expression and the Recurrence Rate for Hematogenous Metastasis by Colorectal Cancer Stage The recurrence rate for hematogenous metastasis was 9.8 % for stage 3 colorectal cancer patients with PROK1 expression–negative primary lesions, whereas it was significantly higher (21.1 %) for patients with stage 3 PROK1 expression–positive lesions. No significant differences were observed between stages 1 and 2 in terms of the relationship between PROK1 expression and recurrence of hematogenous metastasis. The local recurrence rate was 2.1 % for stage 2 colorectal cancer patients with PROK1 expression–negative primary lesions, whereas it was significantly higher (12 %) for patients with stage 2 PROK1 expression–positive lesions (Table 2). Relationship Between PROK1 Expression and Survival Rate by Colorectal Cancer Stage The 5-year survival rate for stage 3 colorectal cancer patients with PROK1 expression–negative primary lesions was 84.8 %, whereas it was 65.2 % for patients with PROK1 expression–positive lesions (Fig. 1c-iii). For stage 4 colorectal cancer patients, the 5-year survival rate was 23.1 % for those with PROK1 expression–negative lesions and 8.5 % for those with PROK1 expression–positive lesions (Fig. 1c-iv). Therefore, for both stages, the survival rates were significantly lower for patients with PROK1 expression. No significant differences in survival were observed between patients with stages 1 and 2 colorectal cancer in terms of PROK1 expression in the primary lesions (Fig. 1c-i, ii). PROK1 as a New Prognostic Factor in Colorectal Cancer Univariate analysis using the Cox proportional hazard model showed significant differences in PROK1 expression, histologic type, serosal invasion, lymph node metastasis, peritoneal metastasis, and hematogenous metastasis. Multivariate analysis showed significant differences in PROK1 expression, histologic type, peritoneal metastasis, and liver metastasis. The hazard ratio for PROK1 expression was 1.928 (Table 3).
T. Nakazawa et al.
A (i)
B (i)
(ii)
kD
(ii)
30
C
(i)
(ii) 100 Survival rate (%)
100 Survival rate (%)
GST+ PROK 1
PROK 1
GST+ PROK 1
PROK 1
10
80 60 P=0.326
40 20
PROK1 positive PROK1 negative
80 60 40
P=0.064
20
PROK1 positive PROK1 negative
0
0 0
20 40 Survival time (months)
60
0
(iii)
20 40 Survival time (months)
60
(iv) 100
Survival rate (%)
Survival rate (%)
100 80 60 P=0.002
40 20 0
PROK1 positive PROK1 negative
0
20 40 Survival time (months)
60
80 60
P=0.002
40 PROK1 positive PROK1 negative
20 0
0
20 40 Survival time (months)
60
FIG. 1 a The prokineticin 1 (PROK1) protein produced in Escherichia coli and immunoreactivity with anti-PROK1 mAb (established by our department). (i) Coomassie blue: purified PROK1 protein and PROK1 Glutathione-S-Transferase (GST) fusion protein. (ii) Western blot of purified PROK1 protein and PROK1 fusion protein. Bands were detected by enhanced chemiluminescence. b PROK1 protein expression was detected in various human primary colorectal cancers by immunohistochemical staining with anti-PROK1 mAb. (i) PROK1
expression was not detected in healthy human colorectal mucosa. (ii) PROK1 expression was detected in primary colorectal cancer lesions. c Relationship between PROK1 expression rates and survival rates in colorectal cancer patients. (i) Stage 1. (ii) Stage 2. (iii) Stage 3. (iv) Stage 4. Patients with PROK1-positive tumors had a significantly poorer prognosis than those with PROK1-negative tumors in stages 3 and 4 (p \ 0.05)
Investigation of Tube Formation With PROK1 Protein
DISCUSSION
When only complete medium was added to the tube formation system, the length of tube formation was 680 lm. In contrast, when PROK1 protein was added to the complete medium, tube formation became significantly longer, up to 1,430 lm. When PROK1 protein and anti-PROK1 mAb were added to complete medium, the extension of tube formation was significantly suppressed, down to 600 lm (Fig. 2).
Molecular biologic studies have been performed to determine the metastatic mechanism of various malignant tumors in the blood, gastrointestinal organs, and lungs, and a number of genes are thought to be involved.19,20 Furthermore, molecular drugs targeting these genes have been developed for potential improvement of the prognosis for cancer patients.21–24 Angiogenic growth factors in partic-
PROK1 as a New Prognostic Factor TABLE 1 Correlation between clinicopathologic findings and prokineticin 1 (PROK1) expression PROK 1-positive
No. of cases
No. of cases
%
All cases
620
223
36.0
TABLE 1 continued PROK 1-positive
p value 4
Gender Male Female
0.827 365 255
130 93
35.6 36.5
108
42
38.9
Age (average 66.5 years) \55
0.126
C55–\65
143
56
39.2
C65 to \75
187
70
37.4
C75
182
55
30.2
Location Right colon
194
55
28.4
Left colon
191
51
26.7
Rectum
235
117
49.7
Histologic type Well?mod
0.321 572
204
35.7
Poor
28
14
50.0
Mucinous
20
5
25.0
99
27
27.3
521
196
37.6
Negative
244
76
31.1
Positive
376
147
39.1
Negative
600
213
35.5
Positive
20
10
50.0
Negative
561
185
33.0
Positive
59
38
64.4
Lymphatic invasion Negative Positive
0.049
Venous invasion
0.043
Peritoneal metastasis
0.184
\0.001
Hematogenous metastasis
T (TNM 6th)
0.001
T1
74
11
14.9
T2
74
28
37.8
T3
223
84
37.7
249
100
40.2
T4 N (TNM 6th)
\0.001
\0.001
N0
329
92
28.0
N1
176
67
38.1
N2
115
64
55.7
1
117
25
21.4
2A
114
34
29.8
2B
85
24
28.2
3A
19
7
36.8
3B
130
46
35.4
3C
74
37
50.0
\0.001
Stage (TNM 6th)
No. of cases 81
No. of cases 50
%
p value
61.7
Well well differentiated adenocarcinoma, mod moderately differentiated adenocarcinoma, poor poorly differentiated adenocarcinoma, mucinous mucinous adenocarcinoma
ular are known to be essential factors for the survival, growth, and development of tumors.3–5 The growth factor examined in the current study (PROK1) was initially cloned as a vascular endothelial growth factor in endocrine cells.9 We found that when the PROK1 gene is introduced into colorectal cancer cells, it promotes hematogenous metastasis and angiogenesis into the surrounding tissue.10 In prostate cancer, neuroblastoma, and pancreatic duct cancer, findings have shown that PROK1 is related to malignancy.12–14 Therefore, its significance for malignant tumors appears to be substantial. However, because no reports on PROK1 protein levels in malignant tumor tissues were available, our department independently developed a mAb for immunohistochemical staining of PROK1 in resected colorectal cancer tissue samples. We aimed to find a new indicator for the growth of colorectal cancer based on the status of PROK1 expression. We observed no PROK1 expression in healthy gastrointestinal mucosa, but PROK1 expression was noted in approximately 40 % of the primary colorectal cancer lesions. Clinicopathologic examination confirmed that PROK1 expression frequently occurred in cases with angiogenic growth-associated vascular invasion and hematogenous metastasis, including liver metastasis. Recent findings suggest that an appropriate environment (niche) is required for the growth of cancer cells and that cytokines, extracellular matrix, angiogenic factors, mesenchymal cells, paraneoplastic macrophages, and fibroblasts are the factors needed for such an environment.25–27 We confirmed that PROK1 protein significantly increased tube formation. Based on the aforementioned findings, colorectal cancer cells are believed to promote PROK1 expression to prepare a suitable environment for cancer cell growth and development. The recurrence rate of stage 3 colorectal cancer is considered to be as high as 20–35 %.1 Therefore, it is important to identify the factors involved in the recurrence of stage 3 cancer to improve therapeutic outcomes. Recurrence should be detected at an early stage through detailed follow-up assessment together with chemotherapy after curative resection to establish a treatment regimen for improved prognosis. Some reports describe the usefulness
T. Nakazawa et al. TABLE 2 Relationship between prokineticin 1 (PROK1) expression and the rate of metastasis recurrence by colorectal cancer stage Stage grouping
PROK 1-negative No. of cases
PROK 1-positive
p value
Recurrence
%
No. of cases
Recurrence
%
Hematogenous metastasis All cases
365
21
5.8
173
25
14.5
0.002
Stage 1
92
1
1.1
25
1
4.0
0.913
Stage 2
141
7
5.0
58
5
8.6
0.357
Stage 3
132
13
9.8
90
19
21.1
0.044
All cases Stage 1
365 92
9 0
2.5 0
173 25
14 0
8.1 0
0.003 –
Stage 2
141
3
2.1
58
7
Stage 3
132
6
4.5
90
7
Local recurrence
12 7.8
0.004 0.314
Peritoneal metastasis All cases
365
4
1.1
173
5
2.9
0.130
Stage 1
92
0
0
25
0
0
–
Stage 2
141
2
1.4
58
2
3.4
0.354
Stage 3
132
2
1.5
90
3
3.3
0.441
Lymphnode metastasis All cases
365
7
1.9
173
7
4.0
0.148
Stage 1
92
0
0
25
0
0
–
Stage 2
141
0
0
58
0
0
–
Stage 3
132
7
5.3
90
7
7.8
0.456
TABLE 3 Pathological findings and prokineticin 1 (PROK1) as a prognostic factor for colorectal cancer patients Univariate analysis
Multivariate analysis
HR
95 % CI
p value
HR
95 % CI
Gender
0.772
0.556–1.072
Age
0.998
0.993–1.003
0.435
PROK1
2.206
1.467–3.318
Histologic type
2.019
1.441–2.830
p value
\0.001
2.109
1.377–3.229
0.001
\0.001
1.928
1.319–2.819
\0.001
0.122
Well?mod/poor/muc Lymphatic invasion
1.684
0.779–3.640
0.185
Venous invasion
1.004
0.648–1.555
0.987
Peritoneal metastasis
7.960
4.023–15.749
\0.001
4.531
2.169–9.466
\0.001
Hematogenous
3.311
2.201–4.981
\0.001
1.701
1.088–2.660
0.019
4.240 2.203
1.597–11.547 1.406–3.450
\0.001 \0.001
2.270 1.447
0.782–6.298 0.899–2.330
0.134 0.128
Metastasis T4 N1 and N2
HR hazard ratio, CI confidence interval, Well well differentiated adenocarcinoma, mod moderately differentiated adenocarcinoma, poor poorly differentiated adenocarcinoma, muc mucinous adenocarcinoma
of the factors, including microsatellite instability MSI as the judgment.28,29 In the current study, recurrence was significantly more frequent and prognosis was worse for the stage 3 patients with PROK1 expression–positive lesions than for stage 3 patients with PROK1 expression–negative lesions. Multivariate analysis using the Cox proportional hazard model
showed PROK1 expression as an independent prognostic factor, indicating that PROK1 expression could be an important factor for the infiltration and metastasis of human colorectal cancer. A recent finding identified PK-R1 and PK-R2 as PROK1 protein receptors in colon cancer cells and showed that the cell infiltration capability of PROK1 is intensified via the
PROK1 as a New Prognostic Factor FIG. 2 Investigation of tube formation with prokineticin 1 (PROK1) protein. a Representative photographs of tube formation treated with PROK1. The culture medium with PROK1 protein plus anti-PROK1 mAb was added to wells of an angiogenesis kit (Kurabo Co., Neyagawa, Japan). On day 5, cells were fixed in 70 % ethanol and stained with anti-CD31 mAb. Left: Tube formation with PROK1 protein. Right: Tube formation with PROK1 protein plus anti-PROK1 mAb. b Quantitative analyses of the tube length. Tube formation was evaluated by measurements of tube length after treatment with PROK1 protein plus anti-PROK1 mAb. Data represent means ± SEM (n = 4). *p \ 0.05, Student’s t test
A PROK1 protein Anti-PROK1 Ab
– –
+ –
*
B
+ +
*
(µm) 1750 1500 1250 1000 750 500 250 0 PROK1 protein Anti-PROK1 Ab
autocrine mechanism.11 In general, augmentation of infiltration leads to infiltration/metastasis of cancer cells into the surrounding organs,30–32 which supports our clinicopathologic findings. Although this was a retrospective study, the expression of PROK1 protein was identified for the first time as a new prognosis-determining factor in human colorectal cancer using an antibody developed in our department. ACKNOWLEDGMENT The technical assistance of Ms. M. Saitoh with this research was appreciated. This work was supported in part by a Grant-in-Aid for Science Research(C) from the Ministry of Education, Sports, Science and Technology of Japan (No. 25462047). CONFLICT OF INTEREST have nothing to disclose.
Nakazawa, Goi, Hirono, Yamaguchi
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ORIGINAL ARTICLE – COLORECTAL CANCER
Prokineticin 1 Protein Expression is a Useful New Prognostic Factor for Human Sporadic Colorectal Cancer Toshiyuki Nakazawa, MD, Takanori Goi, MD, PhD, Yasuo Hirono, MD, PhD, and Akio Yamaguchi, MD, PhD First Department of Surgery, University of Fukui, Fukui, Japan
ABSTRACT Background. Hematogenous metastasis, regarded as closely related to angiogenic growth factors, is associated with colorectal cancer prognosis. The angiogenic growth factor prokineticin 1 (PROK1) has been cloned from endocrine cells. However, its protein expression in human malignant tumors has not been studied. The current study established the anti-PROK1 monoclonal antibody (mAb) and examined the relationship between the expression of PROK1 protein and human colorectal cancer. Methods. The expression of PROK1 protein was assessed in 620 resected sporadic colorectal cancer tissue samples by immunohistochemical staining with in-house-developed human PROK1 mAb to investigate the relationship of PROK1 expression to clinicopathologic factors, recurrence, and survival rate and to evaluate its prognostic significance. Results. The expression of PROK1 protein was detected in 36 % (223/620) of human primary colorectal cancer lesions but no in the healthy mucosa adjacent to the colorectal cancer lesions. According to the clinicopathologic examinations, the frequency of positive PROK1 expression was significantly higher in cases with serosal invasion, lymphatic invasion, venous invasion, lymph node metastasis, liver metastasis, hematogenous metastasis, and higher stage disease. The recurrence rate and prognosis for patients with PROK1 expression–positive lesions were significantly worse. In the Cox proportional hazard model, PROK1 expression was an independent prognostic factor. Conclusions. The expression of PROK1 protein was identified for the first time as a new prognostic factor in colorectal cancer.
Ó Society of Surgical Oncology 2014 First Received: 14 May 2014 T. Goi, MD, PhD e-mail: [email protected]
To improve prognosis in colorectal cancer treatment, measures taken against hepatic and other hematogenous metastases are of the utmost importance.1 Such measures include identification of patients at risk for hematogenous metastasis so that hematogenous metastases can be detected early and treated promptly with intensive postoperative adjuvant therapy and follow-up observation. According to a recently proposed hypothesis on the mechanism of colorectal cancer metastasis, tumor cells first detach from the primary lesion. This detachment is followed by disintegration of the basement membrane, migration of the tumor cells into the interstitium, and vascular invasion.2 A number of studies have reported many genes that might be involved in each step of metastasis based on molecular biologic analyses.3–5 Angiogenic growth factors are known to be important, and several angiogenic growth factors are thought to be involved in the development of colorectal cancer.6–8 Prokineticin 1 (PROK1), the protein examined in the current study, was first reported as an angiogenic growth factor by Ferrara.9 Analysis of PROK1 mRNA expression in healthy tissues showed that it is expressed in limited tissue types including the adrenal gland, ovary, testis, and other endocrine tissues.9 Vascular endothelial growth is promoted by PROK1 after its induction under hypoxic conditions, but growth activity and chemotactic activity are observed only in endocrine cells.9 We recently reported that the introduction of PROK1 into a colorectal cancer cell line frequently induces angiogenesis and hematogenous metastasis10 and that cellular infiltration is promoted by the autocrine mechanism.11 Other reports on PROK1 and malignant tumors have shown that PROK1 is related to prostate cancer and neuroblastoma metastasis and that it increases the malignancy of pancreatic duct cancer, suggesting that PROK1 activity is important in malignant tumors.12–14 Nevertheless, no studies have investigated the expression of PROK1 in human colorectal cancer. We therefore performed a detailed analysis of
T. Nakazawa et al.
PROK1 expression in 620 primary colorectal cancer lesions to assess the relationship between PROK1 expression and clinicopathologic factors, recurrence rate, and prognosis. MATERIALS AND METHODS Construction of Plasmid pGEX-PROK1 and Affinity Purification of Bacterial Extracts The total RNA was extracted from the colon cancer cells using guanidinium-thiocyanate. Single-strand cDNA prepared from 3 lg of total RNA using Moloney murine leukemia virus reverse transcriptase (Gibco-BRL, Rockville, MD, USA) with an oligo (dT) primer-14 was used as the template for the polymerase chain reaction (PCR).15 The products were used for 30 cycles of PCR amplification using two primers: the 50 primer, PROK1-AX, GGATC CATGAGAGGTGCCACGCGAGTCTCAATC of the published human PROK1 sequence9 and the 30 primer, PROK1BX, GAATTCAAAATTGATGTTCTTCAAGTCCATGG AGCAGCGG. Next, 35 cycles of denaturation (94 °C, 1 min), annealing (52 °C, 1.5 min), and extension (72 °C, 2.5 min) were carried out in a thermal cycler (PTC-100, Programmable Thermal Controller; NJ Research Inc., USA). Then, 50 ll of the PCR products that showed the relevant bands in reverse transcriptase (RT)-PCR analysis were visualized in ethidium bromide-stained 1.2 % agarose gels, purified, and cloned into pGEX2T vector (Pharmacia, Uppsala, Sweden).16 The plasmid constructs were confirmed by DNA sequencing. Fusion proteins were induced by adding isopropyl-B-Dthiogalactopyranoside to bacterial culture. These fusion proteins were purified by glutathione Sepharose 4B (Pharmacia, Uppsala, Sweden) affinity column.16 Also, PROK1 proteins were purified using thrombin (Pharmacia, Uppsala, Sweden). Western Blot Analysis For Western blot analysis, 5 lg of purified protein was run on a 10 % sodium dodecyl sulfate (SDS)-polyacrylamide gel and transferred to polyvinylidene difluoride (PVDF) membrane. After the electrophoretic transfer, the membrane was blocked overnight at 4 °C and incubated with the primary antibody. The protein bands were incubated with anti-PROK1 monoclonal antibody (mAb) (established by our department). Density was visualized by enhanced chemiluminescence according to the manufacturer’s instructions (Amersham, Piscataway, NJ, USA). In Vitro Tube Formation Assay The samples (fresh medium containing recombinant PROK1 protein; Shenandoah Company, Warwick,
Philadelphia, USA) (10 ng/ml) or the culture fluid plus mAb or normal mouse immunoglobulin G (IgG; Santa Cruz Biotechnology, USA) were added to wells of an angiogenesis kit (Kurabo Co., Neyagawa, Japan). On day 5, cells were fixed in 70 % ethanol and stained with antiCD31 mAb (Tubule staining kit; Kurabo Co., Neyagawa, Japan). For the evaluation of capillary tube formation (the stained tube-like structures), each well was photographed, and the total tube length was analyzed by the MacSCOPE program (Mitani Company, Fukui, Osaka, Japan).15 The experiments were repeated four times. Patients and Samples Surgical specimens and adjacent normal colorectal tissues were obtained from surgical resections from 620 patients with sporadic primary colorectal cancer in the First Department of Surgery, University of Fukui, Japan between 1990 and 2007. The ages of the 620 patients ranged from 22 to 95 years. Cancerous tissues and corresponding normal tissues were obtained at surgery. According to the tumor-nodemetastasis (TMN) classification,17 117 patients had stage 1 cancer, 199 had stage 2 cancer, 223 had stage 3 cancer, and 81 had stage 4 cancer. Because histopathologic findings varied within the same tumors, the diagnosis was based on the dominant pattern evaluated by two pathologists. All samples were fixed in 10 % paraformaldehyde (pH 6.8) for 24 h and embedded in paraffin. The eligibility criteria for the study specified histopathologic findings confirming primary colorectal cancer, resection of colorectal cancer with extended (D2 or D3) lymph node dissection,18 histologic curative resection (stages 1–3), an Eastern Cooperative Oncology Group performance status (PS) of 0 or 1, no chemotherapy or radiotherapy before surgical resection, 5-fluorouracil-based chemotherapy administered after surgical resection to patients with stage 3 or 4 disease, and no chemotherapy administered to patients with stage 1 or 2 disease after surgical resection. All the patients were followed up for recurrence at regular intervals for 5 years and underwent chest X-ray, computed tomography, and colonoscopy. Immunohistochemical Study Paraffin sections 4 lm thick were deparaffinized with xylene and then dehydrated through a graded ethanol series. Endogenous peroxidase activity was blocked by incubation for 30 min with 1 % hydrogen peroxidase in methanol. These hydrate sections were incubated in a dilution of normal goat serum at room temperature for 20 min to reduce nonspecific staining and then incubated with anti-PROK1 mAb for 1 h. After washing with tris buffered saline, and analyzed for the expression of PROK1 protein by the
PROK1 as a New Prognostic Factor
ChemMate method described earlier, the slides finally were lightly counterstained with hematoxylin. The expression was interpreted as positive when the protein was expressed in more than 30 % of all cancer cells using Image J Software (http://rsb.info.nih.gov/ij/).
clinicopathologic factors showed that gender, age, histologic type, and peritoneal metastasis were not related to PROK1 expression. However, PROK1 expression was observed more frequently in patients with lymphatic invasion, venous invasion, tumor depth, lymph node metastasis, hematogenous metastasis, and TMN stage (Table 1).
Statistical Analysis The association of PROK1 expression with histologic type, primary tumor, serosal invasion, lymphatic invasion, venous invasion, lymph node metastasis, peritoneal metastasis, liver metastasis, and hematogenous metastasis was assessed by cross-tabulation, and statistical significance was determined by the v2 test or Student’s t test using Stat Mate IV (ATMS Co., Ltd., Tokyo, Japan). Life-table analysis was performed using the Kaplan–Meier technique. Life time was overall survival time. The outcomes from different groups of patients were compared by the log rank test using Stat Mate IV (ATMS Co., Ltd., Tokyo, Japan). The Cox proportional hazards model was used in multivariate regression analyses of the survival date using SPSS software (IBMM SPSS Statistics, IBM Corp., Armonk, NY, USA). Differences were considered significant at p values lower than 0.05. RESULTS Development of a Human Monoclonal Anti-PROK1 Antibody After removal of spleen cells from mice immunized with a PROK1-GST fusion protein, the spleen cells were fused with NS-1 myeloma cells. Subcloning and limiting dilution were repeated three times to generate an anti-PROK1 mAb. This antibody detected a protein in the lanes of a gel containing PROK1 and the PROK1-GST fusion (Fig. 1a). PROK1 Protein Expression in Human Colorectal Cancer Primary Lesions and Adjacent Healthy Mucosa The PROK1 protein was expressed in primary colorectal cancer lesions but not in the surrounding human healthy mucosa. A representative example is shown in Fig. 1b. We observed PROK1 protein expression in 223 of the 620 human colorectal cancer cases examined. PROK1 Protein Expression in Human Colorectal Cancer Tissue and the Associated Clinicopathologic Factors Examination of the relationship between PROK1 protein expression in primary colorectal cancer lesions and
Relationship Between PROK1 Expression and the Recurrence Rate for Hematogenous Metastasis by Colorectal Cancer Stage The recurrence rate for hematogenous metastasis was 9.8 % for stage 3 colorectal cancer patients with PROK1 expression–negative primary lesions, whereas it was significantly higher (21.1 %) for patients with stage 3 PROK1 expression–positive lesions. No significant differences were observed between stages 1 and 2 in terms of the relationship between PROK1 expression and recurrence of hematogenous metastasis. The local recurrence rate was 2.1 % for stage 2 colorectal cancer patients with PROK1 expression–negative primary lesions, whereas it was significantly higher (12 %) for patients with stage 2 PROK1 expression–positive lesions (Table 2). Relationship Between PROK1 Expression and Survival Rate by Colorectal Cancer Stage The 5-year survival rate for stage 3 colorectal cancer patients with PROK1 expression–negative primary lesions was 84.8 %, whereas it was 65.2 % for patients with PROK1 expression–positive lesions (Fig. 1c-iii). For stage 4 colorectal cancer patients, the 5-year survival rate was 23.1 % for those with PROK1 expression–negative lesions and 8.5 % for those with PROK1 expression–positive lesions (Fig. 1c-iv). Therefore, for both stages, the survival rates were significantly lower for patients with PROK1 expression. No significant differences in survival were observed between patients with stages 1 and 2 colorectal cancer in terms of PROK1 expression in the primary lesions (Fig. 1c-i, ii). PROK1 as a New Prognostic Factor in Colorectal Cancer Univariate analysis using the Cox proportional hazard model showed significant differences in PROK1 expression, histologic type, serosal invasion, lymph node metastasis, peritoneal metastasis, and hematogenous metastasis. Multivariate analysis showed significant differences in PROK1 expression, histologic type, peritoneal metastasis, and liver metastasis. The hazard ratio for PROK1 expression was 1.928 (Table 3).
T. Nakazawa et al.
A (i)
B (i)
(ii)
kD
(ii)
30
C
(i)
(ii) 100 Survival rate (%)
100 Survival rate (%)
GST+ PROK 1
PROK 1
GST+ PROK 1
PROK 1
10
80 60 P=0.326
40 20
PROK1 positive PROK1 negative
80 60 40
P=0.064
20
PROK1 positive PROK1 negative
0
0 0
20 40 Survival time (months)
60
0
(iii)
20 40 Survival time (months)
60
(iv) 100
Survival rate (%)
Survival rate (%)
100 80 60 P=0.002
40 20 0
PROK1 positive PROK1 negative
0
20 40 Survival time (months)
60
80 60
P=0.002
40 PROK1 positive PROK1 negative
20 0
0
20 40 Survival time (months)
60
FIG. 1 a The prokineticin 1 (PROK1) protein produced in Escherichia coli and immunoreactivity with anti-PROK1 mAb (established by our department). (i) Coomassie blue: purified PROK1 protein and PROK1 Glutathione-S-Transferase (GST) fusion protein. (ii) Western blot of purified PROK1 protein and PROK1 fusion protein. Bands were detected by enhanced chemiluminescence. b PROK1 protein expression was detected in various human primary colorectal cancers by immunohistochemical staining with anti-PROK1 mAb. (i) PROK1
expression was not detected in healthy human colorectal mucosa. (ii) PROK1 expression was detected in primary colorectal cancer lesions. c Relationship between PROK1 expression rates and survival rates in colorectal cancer patients. (i) Stage 1. (ii) Stage 2. (iii) Stage 3. (iv) Stage 4. Patients with PROK1-positive tumors had a significantly poorer prognosis than those with PROK1-negative tumors in stages 3 and 4 (p \ 0.05)
Investigation of Tube Formation With PROK1 Protein
DISCUSSION
When only complete medium was added to the tube formation system, the length of tube formation was 680 lm. In contrast, when PROK1 protein was added to the complete medium, tube formation became significantly longer, up to 1,430 lm. When PROK1 protein and anti-PROK1 mAb were added to complete medium, the extension of tube formation was significantly suppressed, down to 600 lm (Fig. 2).
Molecular biologic studies have been performed to determine the metastatic mechanism of various malignant tumors in the blood, gastrointestinal organs, and lungs, and a number of genes are thought to be involved.19,20 Furthermore, molecular drugs targeting these genes have been developed for potential improvement of the prognosis for cancer patients.21–24 Angiogenic growth factors in partic-
PROK1 as a New Prognostic Factor TABLE 1 Correlation between clinicopathologic findings and prokineticin 1 (PROK1) expression PROK 1-positive
No. of cases
No. of cases
%
All cases
620
223
36.0
TABLE 1 continued PROK 1-positive
p value 4
Gender Male Female
0.827 365 255
130 93
35.6 36.5
108
42
38.9
Age (average 66.5 years) \55
0.126
C55–\65
143
56
39.2
C65 to \75
187
70
37.4
C75
182
55
30.2
Location Right colon
194
55
28.4
Left colon
191
51
26.7
Rectum
235
117
49.7
Histologic type Well?mod
0.321 572
204
35.7
Poor
28
14
50.0
Mucinous
20
5
25.0
99
27
27.3
521
196
37.6
Negative
244
76
31.1
Positive
376
147
39.1
Negative
600
213
35.5
Positive
20
10
50.0
Negative
561
185
33.0
Positive
59
38
64.4
Lymphatic invasion Negative Positive
0.049
Venous invasion
0.043
Peritoneal metastasis
0.184
\0.001
Hematogenous metastasis
T (TNM 6th)
0.001
T1
74
11
14.9
T2
74
28
37.8
T3
223
84
37.7
249
100
40.2
T4 N (TNM 6th)
\0.001
\0.001
N0
329
92
28.0
N1
176
67
38.1
N2
115
64
55.7
1
117
25
21.4
2A
114
34
29.8
2B
85
24
28.2
3A
19
7
36.8
3B
130
46
35.4
3C
74
37
50.0
\0.001
Stage (TNM 6th)
No. of cases 81
No. of cases 50
%
p value
61.7
Well well differentiated adenocarcinoma, mod moderately differentiated adenocarcinoma, poor poorly differentiated adenocarcinoma, mucinous mucinous adenocarcinoma
ular are known to be essential factors for the survival, growth, and development of tumors.3–5 The growth factor examined in the current study (PROK1) was initially cloned as a vascular endothelial growth factor in endocrine cells.9 We found that when the PROK1 gene is introduced into colorectal cancer cells, it promotes hematogenous metastasis and angiogenesis into the surrounding tissue.10 In prostate cancer, neuroblastoma, and pancreatic duct cancer, findings have shown that PROK1 is related to malignancy.12–14 Therefore, its significance for malignant tumors appears to be substantial. However, because no reports on PROK1 protein levels in malignant tumor tissues were available, our department independently developed a mAb for immunohistochemical staining of PROK1 in resected colorectal cancer tissue samples. We aimed to find a new indicator for the growth of colorectal cancer based on the status of PROK1 expression. We observed no PROK1 expression in healthy gastrointestinal mucosa, but PROK1 expression was noted in approximately 40 % of the primary colorectal cancer lesions. Clinicopathologic examination confirmed that PROK1 expression frequently occurred in cases with angiogenic growth-associated vascular invasion and hematogenous metastasis, including liver metastasis. Recent findings suggest that an appropriate environment (niche) is required for the growth of cancer cells and that cytokines, extracellular matrix, angiogenic factors, mesenchymal cells, paraneoplastic macrophages, and fibroblasts are the factors needed for such an environment.25–27 We confirmed that PROK1 protein significantly increased tube formation. Based on the aforementioned findings, colorectal cancer cells are believed to promote PROK1 expression to prepare a suitable environment for cancer cell growth and development. The recurrence rate of stage 3 colorectal cancer is considered to be as high as 20–35 %.1 Therefore, it is important to identify the factors involved in the recurrence of stage 3 cancer to improve therapeutic outcomes. Recurrence should be detected at an early stage through detailed follow-up assessment together with chemotherapy after curative resection to establish a treatment regimen for improved prognosis. Some reports describe the usefulness
T. Nakazawa et al. TABLE 2 Relationship between prokineticin 1 (PROK1) expression and the rate of metastasis recurrence by colorectal cancer stage Stage grouping
PROK 1-negative No. of cases
PROK 1-positive
p value
Recurrence
%
No. of cases
Recurrence
%
Hematogenous metastasis All cases
365
21
5.8
173
25
14.5
0.002
Stage 1
92
1
1.1
25
1
4.0
0.913
Stage 2
141
7
5.0
58
5
8.6
0.357
Stage 3
132
13
9.8
90
19
21.1
0.044
All cases Stage 1
365 92
9 0
2.5 0
173 25
14 0
8.1 0
0.003 –
Stage 2
141
3
2.1
58
7
Stage 3
132
6
4.5
90
7
Local recurrence
12 7.8
0.004 0.314
Peritoneal metastasis All cases
365
4
1.1
173
5
2.9
0.130
Stage 1
92
0
0
25
0
0
–
Stage 2
141
2
1.4
58
2
3.4
0.354
Stage 3
132
2
1.5
90
3
3.3
0.441
Lymphnode metastasis All cases
365
7
1.9
173
7
4.0
0.148
Stage 1
92
0
0
25
0
0
–
Stage 2
141
0
0
58
0
0
–
Stage 3
132
7
5.3
90
7
7.8
0.456
TABLE 3 Pathological findings and prokineticin 1 (PROK1) as a prognostic factor for colorectal cancer patients Univariate analysis
Multivariate analysis
HR
95 % CI
p value
HR
95 % CI
Gender
0.772
0.556–1.072
Age
0.998
0.993–1.003
0.435
PROK1
2.206
1.467–3.318
Histologic type
2.019
1.441–2.830
p value
\0.001
2.109
1.377–3.229
0.001
\0.001
1.928
1.319–2.819
\0.001
0.122
Well?mod/poor/muc Lymphatic invasion
1.684
0.779–3.640
0.185
Venous invasion
1.004
0.648–1.555
0.987
Peritoneal metastasis
7.960
4.023–15.749
\0.001
4.531
2.169–9.466
\0.001
Hematogenous
3.311
2.201–4.981
\0.001
1.701
1.088–2.660
0.019
4.240 2.203
1.597–11.547 1.406–3.450
\0.001 \0.001
2.270 1.447
0.782–6.298 0.899–2.330
0.134 0.128
Metastasis T4 N1 and N2
HR hazard ratio, CI confidence interval, Well well differentiated adenocarcinoma, mod moderately differentiated adenocarcinoma, poor poorly differentiated adenocarcinoma, muc mucinous adenocarcinoma
of the factors, including microsatellite instability MSI as the judgment.28,29 In the current study, recurrence was significantly more frequent and prognosis was worse for the stage 3 patients with PROK1 expression–positive lesions than for stage 3 patients with PROK1 expression–negative lesions. Multivariate analysis using the Cox proportional hazard model
showed PROK1 expression as an independent prognostic factor, indicating that PROK1 expression could be an important factor for the infiltration and metastasis of human colorectal cancer. A recent finding identified PK-R1 and PK-R2 as PROK1 protein receptors in colon cancer cells and showed that the cell infiltration capability of PROK1 is intensified via the
PROK1 as a New Prognostic Factor FIG. 2 Investigation of tube formation with prokineticin 1 (PROK1) protein. a Representative photographs of tube formation treated with PROK1. The culture medium with PROK1 protein plus anti-PROK1 mAb was added to wells of an angiogenesis kit (Kurabo Co., Neyagawa, Japan). On day 5, cells were fixed in 70 % ethanol and stained with anti-CD31 mAb. Left: Tube formation with PROK1 protein. Right: Tube formation with PROK1 protein plus anti-PROK1 mAb. b Quantitative analyses of the tube length. Tube formation was evaluated by measurements of tube length after treatment with PROK1 protein plus anti-PROK1 mAb. Data represent means ± SEM (n = 4). *p \ 0.05, Student’s t test
A PROK1 protein Anti-PROK1 Ab
– –
+ –
*
B
+ +
*
(µm) 1750 1500 1250 1000 750 500 250 0 PROK1 protein Anti-PROK1 Ab
autocrine mechanism.11 In general, augmentation of infiltration leads to infiltration/metastasis of cancer cells into the surrounding organs,30–32 which supports our clinicopathologic findings. Although this was a retrospective study, the expression of PROK1 protein was identified for the first time as a new prognosis-determining factor in human colorectal cancer using an antibody developed in our department. ACKNOWLEDGMENT The technical assistance of Ms. M. Saitoh with this research was appreciated. This work was supported in part by a Grant-in-Aid for Science Research(C) from the Ministry of Education, Sports, Science and Technology of Japan (No. 25462047). CONFLICT OF INTEREST have nothing to disclose.
Nakazawa, Goi, Hirono, Yamaguchi
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