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Kindlin-2: a novel adhesion protein related to tumor invasion, lymph node metastasis, and patient outcome in gastric cancer
The American Journal of Surgery (2012) 203, 222–229
Scientific (Exp)/Research
Kindlin-2: a novel adhesion protein related to tumor invasion, lymph node metastasis, and patient outcome in gastric cancer Zhanlong Shen, M.D., Ph.D.a,b, Yingjiang Ye, M.D., Ph.D.a, Lingyi Dong, Ph.D.a, Sanna Vainionpää, B.Sc.b, Harri Mustonen, Ph.D.b, Pauli Puolakkainen, M.D., Ph.D.b, Shan Wang, M.D., Ph.D.a,* a
Department of Gastroenterological Surgery, Peking University, People’s Hospital, Beijing 100044, China; bDepartment of Surgery, Helsinki University Central Hospital, University of Helsinki, Helsinki, Finland KEYWORDS: Kindlin-2; Gastric cancer; Invasion; Metastasis; Prognosis; Therapy
Abstract BACKGROUND: Kindlin-2 has been confirmed as an essential element of bidirectional integrin signaling. In recent years, the relationship between Kindlin-2 expression and cancers has been a focus of interest. However, the relationship between Kindlin-2 expression in gastric cancer and tumor invasion, metastasis, and the outcome of patients have not been studied. METHODS: Kindlin-2 expression at protein and RNA levels were detected by Western blot and real-time polymerase chain reaction in 40 pairs of gastric cancer samples. In addition, the correlations between Kindlin-2 expression and clinicopathologic factors as well as the prognosis of the patients were analyzed. Multivariate Cox regression was used to study the effect of Kindlin-2 expression on overall and progression-free survival. RESULTS: We found that Kindlin-2 was up-regulated both at RNA (P ⫽ .027) and protein levels (P ⫽ .014) in gastric cancer tissues. Tumor samples with high Kindlin-2 expression (Kindlin-2/actin:tumor tissue/paraneoplastic tissue, ⱖ2) was observed in 55% of the patients. Moreover, Kindlin-2 expression had a significant positive correlation with tumor stromal invasion (P ⫽ .014), lymph node metastasis (P ⫽ .007), and TNM stage (P ⫽ .014). Patients with high Kindlin-2 expression had significantly poorer overall survival (P ⫽ .012) and progression-free survival (P ⫽ .012). High Kindlin-2 expression was an independent risk factor of progression-free survival (hazard ratio, 5.2; 95% confidence interval, 1.1–3.3; P ⫽ .032). CONCLUSIONS: Kindlin-2 may play an important role in the development of gastric cancer and it is a potential factor that could be used to evaluate the outcome of gastric cancer. Kindlin-2 may shed new light on evaluating the prognosis and targeted therapy of gastric cancer. Crown Copyright © 2012 Published by Elsevier Inc. All rights reserved.
Zhanlong Shen and Yingjiang Ye contributed equally. * Corresponding author. Tel.: ⫹86-10-88326608; fax: ⫹86-1068792779. E-mail address: [email protected] Manuscript received April 3, 2011; revised manuscript June 3, 2011
Kindlin-2 is a member of Kindlin protein family, which includes Kindlin-1, Kindlin-2, and Kindlin-3.1 Members of the Kindlin family show identical domain structure and high sequence similarities. Human kindlin-2 is approximately 65% and kindlin-3 is approximately 59% identical to human Kindlin-1 at the amino-acid sequence level.2 All the Kindlin
0002-9610/$ - see front matter Crown Copyright © 2012 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.amjsurg.2011.06.050
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family proteins contain a founding members: band 4.1, ezrin, radixin, and moesin (FERM) domain located at the carboxyl terminus, which is an important structural feature because talin and most other FERM-domain– containing proteins have FERM domains in the N-terminal region.3 It has been shown that the Kindlin family’s FERM domains could interact directly with cytoplasmic tails of 1-integrins and 3-integrins.4 – 6 The Kindlin family is considered an adaptor protein recruited to integrin-containing adhesion sites, termed focal adhesions.7–9 Integrins play an important role in mediating adhesion between cells and extracellular matrix (ECM), which is essential for embryonic development and for the maintenance of tissue integrity in adults. Kindlin-1 is expressed in epithelioid cells and loss of Kindlin-1 in human beings can cause Kindler syndrome, with clinical features that include congenital skin blistering and poikiloderma with extensive atrophy.10 Kindlin-3 is expressed in pulmonary and hematopoietic tissues. Mice lacking Kindlin-3 show severe osteopetrosis, hemorrhage, and defects in the erythrocyte membrane skeleton, and died within 1 week of birth.6,11 Kindlin-2 has been detected in many cell types, including fibroblasts, muscle cells, endothelial cells, and epithelial cells.12,13 Kindlin-2 is localized to cell–ECM adhesion sites, and has been shown to interact directly with cytoplasmic tail of 3-integrin. Kindlin-2 overexpression has been shown to induce a modest activation of the ␣IIb3 integrin.14 Kindlin-2 has been found to play a role in embryonic development as well as cardiac development and function. Knockdown of Kindlin-2 in zebra fish induced abnormalities in cardiac structure and function because Kindlin-2– deficient zebra fish displayed ventricular hypoplasia, abnormalities in ventricular contractility, and abnormal cardiac morphology.15 Integrin and associated cell–ECM adhesion proteins play important roles in the regulation of cancer cell invasion.14 Although Kindlin-2 has been confirmed as an essential element of bidirectional integrin signaling,5 little is known about the expression of Kindlin-2 in association with human cancer. Recent studies have shown that Kindlin-2 was expressed differently in breast cancer, uterine leiomyosarcoma, and malignant mesothelioma.16 –18 Gozgit et al17 found that Kindlin-2 was highly expressed in aggressive human TMX2-28 breast cancer cells. Furthermore, suppression of Kindlin-2 expression with small interfering RNA significantly reduced the invasion of TMX2-28 cells through Matrigel (Discovery Labware, Bedford, MA), which indicated that Kindlin-2 might play a positive role in promoting the invasion of breast cancer cells. But Shi and Wu18 drew a different conclusion, they found that Kindlin-2 was expressed abundantly in the SK-LMS-1 leiomyosarcoma cell line, however, Kindlin-2 had a lower expression in the more invasive HT-1080 fibrosarcoma cell line. Moreover, overexpression of Kindlin-2 in the HT-1080 and SKLMS-1 cell lines substantially reduced their ability to in-
223 vade matrigel in the matrigel invasion assay. Therefore, the role of Kindlin-2 may vary between cancer types. Gastric cancer remains the fourth most common malignancy in the world, with an estimated 1 million new cases per year.19 Globally, it is the second leading cause of cancer-related death in men and the fourth among women.19 However, the molecular basis of the origin and development of gastric cancer is still obscure. The function of Kindlin-2 as a novel protein related to cell–ECM adhesion in gastric cancer, as well as the relationship between Kindlin-2 and clinicopathologic factors and the prognosis of patients, to our knowledge has not been studied yet. In our study, we have investigated the expression of Kindlin-2 in a series of gastric cancer tissue and paired paraneoplastic tissue by Western blot and real-time polymerase chain reaction (PCR). We have studied the relationship between Kindlin-2 expression, clinicopathologic factors, and prognosis, which may shed new light on evaluating the prognosis and targeted therapy of gastric cancer.
Materials and Methods Materials Samples including the gastric carcinoma and corresponding paraneoplastic tissue were obtained from 40 consecutive patients with primary gastric cancer who had undergone gastrectomy at the Department of Gastroenterological Surgery at Peking University People’s Hospital between 2001 and 2004. Informed consent was obtained from each patient participating the study. The samples were collected to liquid nitrogen in 5 minutes after surgical resection, and then stored in a ⫺80°C freezer until used. The age range of the subjects was 47 to 93 years, with a mean age of 67.1 ⫾ 12.8 years. Clinicopathologic data are shown in Table 1. All patients received 5-fluorouracil– based chemotherapy postoperatively. None of the patients received chemotherapy and radiotherapy preoperatively.
Methods Western-blot analysis. Gastric cancer and corresponding paraneoplastic tissues or gastric cancer cells were lysed in radioimmunoprecipitation assay buffer (50 mmol/L Tris-Cl, 150 mmol/L NaCl, 1% NP-40, .5% sodium deoxycholate, and .1% sodium dodecyl sulfate, pH 7.5) containing 1 mmol/L phenylmethylsulfonyl fluoride for 30 minutes on ice. The samples were centrifuged and the protein concentrations for each sample were determined by using the Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA). The protein samples (20 g) were separated by 10% sodium dodecylsulfate–polyacrylamide gel electrophoresis, and then electrotransferred to a ni-
224 Table 1
The American Journal of Surgery, Vol 203, No 2, February 2012 Clinicopathologic data of 40 cases
Type Age ⱕ60 y ⬎60 y Sex Male Female Tumor differentiation Well Moderately Poorly Pathologic stage I–II III–IV Tumor invasion depth T1–2 T3–4 Lymph node status N1 N2 N3 Distant metastasis Negative Positive
Cases (n ⫽ 40)
Percent
14 26
35.0 65.0
30 10
75.0 25.0
4 8 28
10.0 20.0 70.0
8 32
20.0 80.0
8 32
20.0 80.0
21 10 9
52.5 25.0 22.5
37 3
92.5 7.5
trocellulose membrane. After blocking with 5% nonfat dry milk in Tween– TRIS-buffered saline solution (.1% Tween-20 in 100 mmol/L Tris-HCl [pH 7.5], .9% NaCl), the membranes were incubated with rabbit monoclonal antibody against human Kindlin-2 (ab74030; Abcam, Cambridge, UK) at a dilution of 1:600 at 4°C overnight. After washings, the blots were incubated with horseradish-peroxidase– conjugated secondary goat anti-rabbit immunoglobulin G antibodies at a dilution of 1:10,000 for 1 hour. The blot was washed and developed using chemiluminescence (Santa Cruz Biotechnology, Santa Cruz, CA). For control, we detected -actin with primary mouse monoclonal antibodies against -actin (sc-47778; Santa Cruz Biotechnology) and densitometric data of Kindlin-2 were normalized to those of -actin levels. Based on the ratio of gray values, Kindlin-2 expression was considered high if the value of Kindlin-2/-actin of tumor was more than 2 times higher than that of paraneoplastic tissue, otherwise it was considered as low expression. Real-time quantitative PCR. Total RNA was extracted from gastric cancer and corresponding paraneoplastic tissues or gastric cancer cells by using TRIzol (Invitrogen Life Technologies, Carlsbad, CA) and quantified by absorbance at 260 nm. The integrity and relative quantity of each RNA sample was confirmed using a miniformaldehyde gel electrophoresis. Reverse transcription was performed at 37°C for 60 minutes with the Quantscript RT Kit (KR103; Tiangen Biotechnology, Beijing, China), 2 g total RNA was used.
All samples were quantified after PCR amplification using a RealMasterMix (SYBR Green) kit (FP202; Tiangen Biotechnology). We used the following set of primers: forward primer Kindlin-2-F, 5-AAATGGTCACCGTAGAGTTTGC-3; reverse primer Kindlin-2-R, 5-CTCTCGTTTTGGTCTTTTGCAC-3. -actin primers used the following set: forward primer, 5-AAGGTGACAGCAGTCGGTT-3; reverse primer, 5-AAGTGGGGTGGCTTTTAG-3. The PCR protocol was as follows: initial denaturation at 95°C for 10 minutes, followed by 30 cycles at 95°C for 10 seconds, annealing at 58.5°C for 5 seconds, and extension at 72°C for 8 seconds. PCR products were quantified via molten curve at 84°C. All results were normalized to -actin to ensure a uniform amount of RNA template. Statistical analysis. The Statistical Package for Social Science (SPSS, Chicago, IL) version 17.0 was used. A nonparametric test was used to compare Kindlin-2 expression in tumor tissues and paraneoplastic tissues. Differences of Kindlin-2 expression and clinical and pathologic variables were compared using the Fisher exact test or the Cochran–Armitage trend test in the case of ordered categories. Overall survival time was calculated from the time since surgery to the last follow-up evaluation or date of death. The median follow-up time for survival was 37.1 months (range, 5–77 mo). Progression-free survival was calculated from the time of surgery to the time of first recurrence and the time from surgery to the last follow-up evaluation/death for patients who did not recur. Univariate survival analysis (overall and progression free) was estimated using Kaplan–Meier methods, and survival time was compared using the log-rank test. The Cox proportional hazards model was used for multivariate analysis. P values were considered significant at a P value of less than .05.
Results Kindlin-2 expression is high at protein and RNA levels in gastric cancer tissues We characterized the expression of Kindlin-2 in 40 paired gastric cancer and paraneoplastic tissues by Western blot. The results were measured by the ratio of Kindlin-2 and -actin gray values. We found that the mean value of Kindlin-2/-actin was higher in gastric cancer tissues than in paraneoplastic tissues (.413 ⫾ .366 and .133 ⫾ .186, respectively; P ⫽ .014; Fig. 1). High expression of Kindlin-2 (normalized tumor expression/normalized paraneoplastic expression, ⬎2) was found in 22 tumors (55% of patients), whereas relative low (normalized tumor expression/normalized paraneoplastic expression ⱕ2) expression was found in 18 tumors (45% of cases). Messenger RNA (mRNA) expression levels of Kindlin-2 was detected by real-time PCR. We found that
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Figure 1 (A) Expression of Kindlin-2 at the protein level in tumor and normal tissues. Western blot samples were made from tumor tissues of gastric cancer patients and normal paraneoplastic tissues of the same patients. Western blot gray values were normalized with -actin gray values of the same samples. The results show an increased protein level expression of Kindlin-2 in tumor tissues (the result was mean ⫾ standard deviation, n ⫽ 40). (B) Expression of Kindlin-2 at the protein level in gastric cancer tissues and paraneoplastic tissues (Western blot). T ⫽ gastric cancer tissue; N ⫽ paraneoplastic tissue.
Kindlin-2 mRNA was detectable in all corresponding paraneoplastic and gastric cancer tissues, but the levels of expression in gastric cancer tissues were much higher than corresponding paraneoplastic tissues (Kindlin-2/actin mRNA expression: 1.02 ⫾ 0.17 vs 0.75 ⫾ .19; P ⫽ .027; Fig. 2).
Kindlin-2 protein expression is related to TNM stage, invasion depth, and lymph node metastasis in gastric cancer There was no correlation between Kindlin-2 protein expression levels (high vs low) and age, sex, or tumor differentiation. There was increased Kindlin-2 protein expression in patients with poor TNM stage, deeper invasion depth, and more lymph node metastasis. Kindlin-2 protein expression levels in locally invasive T3 and T4 tumors were significantly higher when compared with those in less aggressive T1 and T2 tumors (P ⫽ .014).
Kindlin-2 expression levels in Stage II through IV tumors also were significantly higher compared with the expression levels in stages 0 and I tumors (P ⫽ .014). Kindlin-2 expression levels in patients who had more lymph node metastasis (N2, N3) were higher compared with that in patients with a fewer lymph node metastasis (P ⫽ .012; Fisher exact test; Table 2) and there was even a trend showing increasing levels of Kindlin-2 expression on tumors as the node status increased (P ⫽ .003, Cochran– Armitage trend test).
High Kindlin-2 protein expression correlates with poor overall survival and progression-free survival The correlation between Kindlin-2 protein expression levels (high/low) and patient survival with gastric cancer
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Figure 2 Expression of Kindlin-2 at the RNA level in the tumor and normal groups. RNA samples were made from tumor tissues of gastric cancer patients and normal paraneoplastic tissues of the same patients. Real-time PCR results were normalized with -actin results of the same samples. The results show an increased RNA level expression of Kindlin-2 in tumor tissues (the result was mean ⫾ standard deviation, n ⫽ 40).
after surgery was investigated with the Kaplan–Meier method (rate of follow-up evaluation is 100%: 40 of 40). Patients who had high Kindlin-2 expression levels had a significantly shorter overall survival (mean survival time, 20.0 ⫾ 3.71 mo) compared with low Kindlin-2 expression (mean survival time, 62.7 ⫾ 10.3 mo; P ⫽ .012; Fig. 3). Length of progression-free survival of patients with high Kindlin-2 expression levels also was shorter compared with patients with low Kindlin-2 expression (mean, 53.9 ⫾ 10.1 vs 11.4 ⫾ 3.52 mo for low and high Kindlin-2 expression levels, respectively; P ⫽ .012; Fig. 4).
Kindlin-2 protein expression is an independent risk factor affecting progression survival, but not overall survival Because of the correlation between Kindlin-2 expression and tumor stage, tumor invasion depth, and lymph node metastasis, therefore, only age, sex, tumor differentiation, and distant metastasis were examined by multivariate analysis for overall survival and progressionfree survival by the Cox proportional hazards model. We found that Kindlin-2 protein expression was not an independent risk factor of overall survival in gastric cancer (Table 3), but was an independent risk factor of worse progression-free survival (hazard ratio, 5.2; 95% confidence interval, 1.1–3.2; P ⫽ .032; Table 4).
Comments The novel adaptor protein Kindlin-2 is essential for integrin-mediated cell–ECM adhesions. It can interact with cytoplasmic tails of -integrin and subsequently binds to migfilin and recruits it to these focal adhesion sites.12 The presence of migfilin in focal adhesions facilitated the accumulation of other migfilin-binding proteins such as vasodilator-stimulated phosphoprotein, a regulator of actin cytoskeletal dynamics, and filamin, an actin binding protein.20 Moreover, Kindling-2 acts as a crucial co-activator in integrin activation. Ma et al21 showed that co-transfection of Kindlin-2 with talin-H results in a synergistic enhancement of integrin ␣IIb3 activation. Furthermore, short interfering RNA knockdown of endogenous Kindlin-2 impaired talin-induced ␣IIb␣3 activation in transfected Chinese hamster ovary cells and inhibited ␣v3-mediated adhesion and migration of endothelial cells. Kindlin-2 is considered a bidirectional integrin signaling protein involved in the activation of integrin adhesion to ECM and subsequently mediating integrin binding to actin cytoskeleton. Integrin functions in cell-to-cell and cell-to-ECM adhesive interactions and transduce signals from the ECM to the cell interior and vice versa (eg, mediating ECM influence on cell growth and differentiation). Because these properties implicate integrin involvement in cell migration, invasion, intravasation and extravasation, and platelet interaction, a role for integrins in tumor growth
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Correlation between Kindlin-2 and clinicopathologic factors Expression of Kindlin-2 [number (%)]
Age, y ⱕ60 ⬎60 Sex Male Female Tumor differentiation Well Moderately Poorly Pathologic stage I–II III–IV Tumor invasion depth T1–2 T3–4 Lymph node status N1 N2 N3 Distant metastasis Negative Positive
Number (n ⫽ 40)
Low expression (n ⫽ 18)
High expression (n ⫽ 22)
14 26
4 (28.6) 14 (53.8)
10 (71.4) 12 (46.2)
30 10
14 (46.7) 4 (40.0)
16 (53.3) 6 (60.0)
4 8 28
3 (75.0) 5 (62.5) 10 (35.7)
1 (25.0) 3 (37.5) 18 (64.3)
8 32
7 (87.5) 11 (34.4)
1 (12.5) 21 (65.6)
P value, Fisher exact test .186 1.000 .234
.014* .014* 8 32
7 (87.5) 11 (34.4)
1 (12.5) 21 (65.6)
21 10 9
14 (66.7) 3 (30.0) 1 (11.1)
7 (33.3) 7 (70.0) 8 (88.9)
37 3
18 (48.6) 0 (0.0)
19 (51.4) 3 (100.0)
.012*
.238
*Significant at the P ⬍ .05 level.
and metastasis is obvious. As the main link between a cell and the ECM, integrin has an essential role in the invasion process. The cellular integrin expression pattern is highly variable between cancer types. Matsuoka et al22 found that integrin ␣21 expression was associated with tumor progression and invasion in gastric cancer. Kindlin-2 as an activator of integrin might be involved in the development of cancers. It is known that dissemination and metastasis are crucial risk factors of poor prognosis in gastric cancer patients.23–26 Peritoneal dissemination is a common reason why gastric cancer cannot be resected.23 Moreover, peritoneal recurrence after curative resection was identified as a major type of recurrence (29%⬃44%).24,25 Lymph node status is an important prognostic factor regarding long-term survival of gastric cancer patients.26 The 5-year survival rate (after surgical treatment) was 86.1% in the patient group with N0 status, whereas survival rates dramatically decreased to 58.1%, 23.3%, and 5.9% in patient groups with N1, N2, and N3 status, respectively.27 Kindlin-2 has been studied in only a few cancers (eg, breast cancer17 and mesenchymal cancer).18 There are no reports on the relationship between Kindlin-2 and clinicopathologic factors of cancer or Kindlin-2 expression in gastric cancer. In our study, we found that most gastric cancer tissues (85%) had higher Kindlin-2 expression as
compared with normal tissues, and 55% of tumors had very high Kindlin-2 expression (gastric cancer tissues/normal tissues, ⱖ2). We further analyzed the relationship between Kindlin-2 protein expression and clinicopathologic factors. An interesting finding was that gastric cancer tissues with high Kindlin-2 protein expression had deeper invasion and more metastases in lymph nodes and poor TNM stage. Therefore, high Kindlin-2 protein expression might be a factor related to more aggressive biological behavior of gastric cancer. In our study, Kindlin-2 protein expression was significantly higher in stages II through IV than in stages 0 and I tumors. Kindlin-2 expression was related to the depth of tumor invasion and lymph node metastasis. There was no statistical difference in Kindlin-2 expression between patients with or without distant metastasis, which may be owing to the low number of patients3 who had distant metastasis. In addition, we found that patients with high Kindlin-2 expression level had poor overall and progression-free survival. Further, Kindlin-2 expression level was an independent risk factor in progression-free survival, but not in overall survival. High Kindlin-2 expression might facilitate integrin-mediated ECM adhesion and integrin–actin cytoskeleton binding, possibly improving the survival and proliferation of tumor cells, but this requires further research. Overall, Kindlin-2 might
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The American Journal of Surgery, Vol 203, No 2, February 2012 Table 3 Multivariate analysis of overall survival for patients with gastric cancer using the Cox model 95.0% confidence interval for HR
Kindlin-2 Sex Age Differentiation Distant metastasis
P value
HR
Lower
Upper
.175 .425 .267 .430 .248
2.832 .595 .482 1.690 4.202
.630 .166 .133 .460 .368
12.736 2.132 1.750 6.219 48.046
Kindlin-2 low expression, male sex, age ⱕ 60 years, well differentiation, and negative distant metastasis were used as reference categories. HR ⫽ hazard ratio.
Figure 3 Gastric cancer patients with high Kindlin-2 expression in tumors show poor overall survival. Kindlin-2 expression was measured by Western blot in tumors of gastric cancer patients and the patients’ survival was followed up for 89 months (Kaplan– Meier curve, log-rank, P ⫽ .012).
be a marker of poor TNM stage and prognosis of gastric cancer and might become a potential new factor used to evaluate the invasion, metastasis, and outcome of gastric cancer. Li et al28 found that the 5-year survival rate in patients with a positive expression of integrin3 mRNA was significantly lower than for those patients with a negative expression of integrin3 mRNA, and considered that integrin3 high expression can synergistically en-
hance tumor angiogenesis, and may play a crucial role in the invasion and metastasis of gastric carcinoma. However, to our knowledge, the role of Kindlin-2 expression in the prognosis of cancer patients is not clear. Further research is needed to discover the molecular biological mechanism of Kindlin-2 expression in gastric cancer invasion and metastasis affecting the outcome of patients. Moreover, most patients with gastric cancer are diagnosed in the advanced stage, surgery can be performed in only 30% of patients, and a relapse frequently occurs.29 Currently, chemotherapeutic regimens are the mainstay of treatment against advanced gastric cancer. Although oxaliplatin, docetaxel, capecitabine, and irinotecan have shown activity in recent phase III trials and increased the armamentarium of treatment options for patients with advanced gastric cancer,30 the median survival in all randomized trials remains unsatisfactory.31,32 Novel treatment options are urgently needed to improve the outcome of patients with advanced gastric cancer. Our study shows that high Kindlin-2 expression leads to poor
Table 4 Multivariate analysis of the progression-free survival for the gastric cancer with Cox model 95.0% confidence interval for HR
Kindlin-2 Sex Age Differentiation Distant metastasis Figure 4 Poor progression-free survival found in gastric cancer patients with high Kindlin-2 expression in tumors. Kindlin-2 expression was measured by Western blot in tumors of gastric cancer patients and the patients’ progression-free survival was followed up for 84 months (Kaplan–Meier curve, log-rank, P ⫽ .003).
P value
HR
Lower
Upper
.032* .679 .619 .628 .068
5.168 .752 .744 1.277 14.866
1.148 .195 .232 .476 .821
3.272 2.900 2.390 3.429 269.031
Kindlin-2 low expression, male sex, age ⱕ 60 years, well differentiation, and negative distant metastasis were used as reference categories. HR ⫽ hazard ratio. *Significant at the P ⬍ .05 level.
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TNM stage and prognosis of patients, although low Kindlin-2 expression has a better prognosis and TNM stage. Therefore, Kindlin-2 might be a candidate target for therapy to change the outcome of patients with advanced gastric cancer.
Acknowledgments The authors would like to thank Ms. Lingyi Dong and Ms. Sanna Vainionpää for excellent technical assistance with the experiment.
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229 13. Gkretsi V, Zhang Y, Tu Y, et al. Physical and functional association of migfilin with cell-cell adhesions. J Cell Sci 2005;15:697–710. 14. Shi X, Ma YQ, Tu Y, et al. The MIG-2/integrin interaction strengthens cell-matrix adhesion and modulates cell motility. J Biol Chem 2007; 282:20455– 66. 15. Dowling JJ, Gibbs E, Russell M, et al. Kindlin-2 is an essential component of intercalated discs and is required for vertebrate cardiac structure and function. Circ Res 2008;102:423–31. 16. An Z, Dobra K, Lock JG, et al. Kindlin-2 is expressed in malignant mesothelioma and is required for tumor cell adhesion and migration. Int J Cancer 2010;127:1999 –2008. 17. Gozgit JM, Pentecost BT, Marconi SA, et al. Use of an aggressive MCF-7 cell line variant, TMX2-28, to study cell invasion in breast cancer. Mol Cancer Res 2006;4:905–13. 18. Shi X, Wu CA. Suppressive role of mitogen inducible gene-2 in mesenchymal cancer cell invasion. Mol Cancer Res 2008;6:715–24. 19. Global cancer facts and figures, 2007. American Cancer Society. Available from: http://www.cancer.org/Research/CancerFactsFigures/ GlobalCancerFactsFigures/global-cancer-facts-figures-2007. 20. Zhang Y, Tu Y, Gkretsi V, et al. Migfilin interacts with vasodilatorstimulated phosphoprotein (VASP) and regulates VASP localization to cell-matrix adhesions and migration. J Biol Chem 2006;281:12397– 407. 21. Ma YQ, Qin J, Wu C, et al. Kindlin-2 (Mig-2): a co-activator of beta3 integrins. J Cell Biol 2008;5:439 – 46. 22. Matsuoka T, Yashiro M, Nishimura S, et al. Increased expression of alpha2beta1-integrin in the peritoneal dissemination of human gastric carcinoma. Int J Mol Med 2000;5:21–5. 23. Dupont JB Jr, Lee JR, Burton GR, et al. Adenocarcinoma of the stomach: review of 1,497 cases. Cancer 1978;41:941–7. 24. D’Angelica M, Gonen M, Brennan MF, et al. Patterns of initial recurrence in completely resected gastric adenocarcinoma. Ann Surg 2004;240:808 –16. 25. Yoo CH, Noh SH, Shin DW, et al. Recurrence following curative resection for gastric carcinoma. Br J Surg 2000;87:236 – 42. 26. Siewert JR, Böttcher K, Stein HJ, et al. Relevant prognostic factors in gastric cancer: ten-year results of the German Gastric Cancer Study. Ann Surg 1998;228:449 – 61. 27. Zhang XF, Huang CM, Lu HS, et al. Surgical treatment and prognosis of gastric cancer in 2,613 patients. World J Gastroenterol 2004;10: 3405– 8. 28. Li SG, Ye ZY, Zhao ZS, et al. Correlation of integrin beta3 mRNA and vascular endothelial growth factor protein expression profiles with the clinicopathological features and prognosis of gastric carcinoma. World J Gastroenterol 2008;21:421–7. 29. Sugano K. Gastric cancer: pathogenesis, screening, and treatment. Gastrointest Endosc Clin N Am 2008;18:513–22. 30. Field K, Michael M, Leong T. Locally advanced and metastatic gastric cancer: current management and new treatment developments. Drugs 2008;68:299 –317. 31. Wöll E, Devries A, Eisterer W, et al. Chemotherapy in gastric cancer. Anticancer Res 2008;28:1213–9. 32. Sasako M. Surgery and adjuvant chemotherapy. Int J Clin Oncol 2008;13:193–5.
Scientific (Exp)/Research
Kindlin-2: a novel adhesion protein related to tumor invasion, lymph node metastasis, and patient outcome in gastric cancer Zhanlong Shen, M.D., Ph.D.a,b, Yingjiang Ye, M.D., Ph.D.a, Lingyi Dong, Ph.D.a, Sanna Vainionpää, B.Sc.b, Harri Mustonen, Ph.D.b, Pauli Puolakkainen, M.D., Ph.D.b, Shan Wang, M.D., Ph.D.a,* a
Department of Gastroenterological Surgery, Peking University, People’s Hospital, Beijing 100044, China; bDepartment of Surgery, Helsinki University Central Hospital, University of Helsinki, Helsinki, Finland KEYWORDS: Kindlin-2; Gastric cancer; Invasion; Metastasis; Prognosis; Therapy
Abstract BACKGROUND: Kindlin-2 has been confirmed as an essential element of bidirectional integrin signaling. In recent years, the relationship between Kindlin-2 expression and cancers has been a focus of interest. However, the relationship between Kindlin-2 expression in gastric cancer and tumor invasion, metastasis, and the outcome of patients have not been studied. METHODS: Kindlin-2 expression at protein and RNA levels were detected by Western blot and real-time polymerase chain reaction in 40 pairs of gastric cancer samples. In addition, the correlations between Kindlin-2 expression and clinicopathologic factors as well as the prognosis of the patients were analyzed. Multivariate Cox regression was used to study the effect of Kindlin-2 expression on overall and progression-free survival. RESULTS: We found that Kindlin-2 was up-regulated both at RNA (P ⫽ .027) and protein levels (P ⫽ .014) in gastric cancer tissues. Tumor samples with high Kindlin-2 expression (Kindlin-2/actin:tumor tissue/paraneoplastic tissue, ⱖ2) was observed in 55% of the patients. Moreover, Kindlin-2 expression had a significant positive correlation with tumor stromal invasion (P ⫽ .014), lymph node metastasis (P ⫽ .007), and TNM stage (P ⫽ .014). Patients with high Kindlin-2 expression had significantly poorer overall survival (P ⫽ .012) and progression-free survival (P ⫽ .012). High Kindlin-2 expression was an independent risk factor of progression-free survival (hazard ratio, 5.2; 95% confidence interval, 1.1–3.3; P ⫽ .032). CONCLUSIONS: Kindlin-2 may play an important role in the development of gastric cancer and it is a potential factor that could be used to evaluate the outcome of gastric cancer. Kindlin-2 may shed new light on evaluating the prognosis and targeted therapy of gastric cancer. Crown Copyright © 2012 Published by Elsevier Inc. All rights reserved.
Zhanlong Shen and Yingjiang Ye contributed equally. * Corresponding author. Tel.: ⫹86-10-88326608; fax: ⫹86-1068792779. E-mail address: [email protected] Manuscript received April 3, 2011; revised manuscript June 3, 2011
Kindlin-2 is a member of Kindlin protein family, which includes Kindlin-1, Kindlin-2, and Kindlin-3.1 Members of the Kindlin family show identical domain structure and high sequence similarities. Human kindlin-2 is approximately 65% and kindlin-3 is approximately 59% identical to human Kindlin-1 at the amino-acid sequence level.2 All the Kindlin
0002-9610/$ - see front matter Crown Copyright © 2012 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.amjsurg.2011.06.050
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family proteins contain a founding members: band 4.1, ezrin, radixin, and moesin (FERM) domain located at the carboxyl terminus, which is an important structural feature because talin and most other FERM-domain– containing proteins have FERM domains in the N-terminal region.3 It has been shown that the Kindlin family’s FERM domains could interact directly with cytoplasmic tails of 1-integrins and 3-integrins.4 – 6 The Kindlin family is considered an adaptor protein recruited to integrin-containing adhesion sites, termed focal adhesions.7–9 Integrins play an important role in mediating adhesion between cells and extracellular matrix (ECM), which is essential for embryonic development and for the maintenance of tissue integrity in adults. Kindlin-1 is expressed in epithelioid cells and loss of Kindlin-1 in human beings can cause Kindler syndrome, with clinical features that include congenital skin blistering and poikiloderma with extensive atrophy.10 Kindlin-3 is expressed in pulmonary and hematopoietic tissues. Mice lacking Kindlin-3 show severe osteopetrosis, hemorrhage, and defects in the erythrocyte membrane skeleton, and died within 1 week of birth.6,11 Kindlin-2 has been detected in many cell types, including fibroblasts, muscle cells, endothelial cells, and epithelial cells.12,13 Kindlin-2 is localized to cell–ECM adhesion sites, and has been shown to interact directly with cytoplasmic tail of 3-integrin. Kindlin-2 overexpression has been shown to induce a modest activation of the ␣IIb3 integrin.14 Kindlin-2 has been found to play a role in embryonic development as well as cardiac development and function. Knockdown of Kindlin-2 in zebra fish induced abnormalities in cardiac structure and function because Kindlin-2– deficient zebra fish displayed ventricular hypoplasia, abnormalities in ventricular contractility, and abnormal cardiac morphology.15 Integrin and associated cell–ECM adhesion proteins play important roles in the regulation of cancer cell invasion.14 Although Kindlin-2 has been confirmed as an essential element of bidirectional integrin signaling,5 little is known about the expression of Kindlin-2 in association with human cancer. Recent studies have shown that Kindlin-2 was expressed differently in breast cancer, uterine leiomyosarcoma, and malignant mesothelioma.16 –18 Gozgit et al17 found that Kindlin-2 was highly expressed in aggressive human TMX2-28 breast cancer cells. Furthermore, suppression of Kindlin-2 expression with small interfering RNA significantly reduced the invasion of TMX2-28 cells through Matrigel (Discovery Labware, Bedford, MA), which indicated that Kindlin-2 might play a positive role in promoting the invasion of breast cancer cells. But Shi and Wu18 drew a different conclusion, they found that Kindlin-2 was expressed abundantly in the SK-LMS-1 leiomyosarcoma cell line, however, Kindlin-2 had a lower expression in the more invasive HT-1080 fibrosarcoma cell line. Moreover, overexpression of Kindlin-2 in the HT-1080 and SKLMS-1 cell lines substantially reduced their ability to in-
223 vade matrigel in the matrigel invasion assay. Therefore, the role of Kindlin-2 may vary between cancer types. Gastric cancer remains the fourth most common malignancy in the world, with an estimated 1 million new cases per year.19 Globally, it is the second leading cause of cancer-related death in men and the fourth among women.19 However, the molecular basis of the origin and development of gastric cancer is still obscure. The function of Kindlin-2 as a novel protein related to cell–ECM adhesion in gastric cancer, as well as the relationship between Kindlin-2 and clinicopathologic factors and the prognosis of patients, to our knowledge has not been studied yet. In our study, we have investigated the expression of Kindlin-2 in a series of gastric cancer tissue and paired paraneoplastic tissue by Western blot and real-time polymerase chain reaction (PCR). We have studied the relationship between Kindlin-2 expression, clinicopathologic factors, and prognosis, which may shed new light on evaluating the prognosis and targeted therapy of gastric cancer.
Materials and Methods Materials Samples including the gastric carcinoma and corresponding paraneoplastic tissue were obtained from 40 consecutive patients with primary gastric cancer who had undergone gastrectomy at the Department of Gastroenterological Surgery at Peking University People’s Hospital between 2001 and 2004. Informed consent was obtained from each patient participating the study. The samples were collected to liquid nitrogen in 5 minutes after surgical resection, and then stored in a ⫺80°C freezer until used. The age range of the subjects was 47 to 93 years, with a mean age of 67.1 ⫾ 12.8 years. Clinicopathologic data are shown in Table 1. All patients received 5-fluorouracil– based chemotherapy postoperatively. None of the patients received chemotherapy and radiotherapy preoperatively.
Methods Western-blot analysis. Gastric cancer and corresponding paraneoplastic tissues or gastric cancer cells were lysed in radioimmunoprecipitation assay buffer (50 mmol/L Tris-Cl, 150 mmol/L NaCl, 1% NP-40, .5% sodium deoxycholate, and .1% sodium dodecyl sulfate, pH 7.5) containing 1 mmol/L phenylmethylsulfonyl fluoride for 30 minutes on ice. The samples were centrifuged and the protein concentrations for each sample were determined by using the Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA). The protein samples (20 g) were separated by 10% sodium dodecylsulfate–polyacrylamide gel electrophoresis, and then electrotransferred to a ni-
224 Table 1
The American Journal of Surgery, Vol 203, No 2, February 2012 Clinicopathologic data of 40 cases
Type Age ⱕ60 y ⬎60 y Sex Male Female Tumor differentiation Well Moderately Poorly Pathologic stage I–II III–IV Tumor invasion depth T1–2 T3–4 Lymph node status N1 N2 N3 Distant metastasis Negative Positive
Cases (n ⫽ 40)
Percent
14 26
35.0 65.0
30 10
75.0 25.0
4 8 28
10.0 20.0 70.0
8 32
20.0 80.0
8 32
20.0 80.0
21 10 9
52.5 25.0 22.5
37 3
92.5 7.5
trocellulose membrane. After blocking with 5% nonfat dry milk in Tween– TRIS-buffered saline solution (.1% Tween-20 in 100 mmol/L Tris-HCl [pH 7.5], .9% NaCl), the membranes were incubated with rabbit monoclonal antibody against human Kindlin-2 (ab74030; Abcam, Cambridge, UK) at a dilution of 1:600 at 4°C overnight. After washings, the blots were incubated with horseradish-peroxidase– conjugated secondary goat anti-rabbit immunoglobulin G antibodies at a dilution of 1:10,000 for 1 hour. The blot was washed and developed using chemiluminescence (Santa Cruz Biotechnology, Santa Cruz, CA). For control, we detected -actin with primary mouse monoclonal antibodies against -actin (sc-47778; Santa Cruz Biotechnology) and densitometric data of Kindlin-2 were normalized to those of -actin levels. Based on the ratio of gray values, Kindlin-2 expression was considered high if the value of Kindlin-2/-actin of tumor was more than 2 times higher than that of paraneoplastic tissue, otherwise it was considered as low expression. Real-time quantitative PCR. Total RNA was extracted from gastric cancer and corresponding paraneoplastic tissues or gastric cancer cells by using TRIzol (Invitrogen Life Technologies, Carlsbad, CA) and quantified by absorbance at 260 nm. The integrity and relative quantity of each RNA sample was confirmed using a miniformaldehyde gel electrophoresis. Reverse transcription was performed at 37°C for 60 minutes with the Quantscript RT Kit (KR103; Tiangen Biotechnology, Beijing, China), 2 g total RNA was used.
All samples were quantified after PCR amplification using a RealMasterMix (SYBR Green) kit (FP202; Tiangen Biotechnology). We used the following set of primers: forward primer Kindlin-2-F, 5-AAATGGTCACCGTAGAGTTTGC-3; reverse primer Kindlin-2-R, 5-CTCTCGTTTTGGTCTTTTGCAC-3. -actin primers used the following set: forward primer, 5-AAGGTGACAGCAGTCGGTT-3; reverse primer, 5-AAGTGGGGTGGCTTTTAG-3. The PCR protocol was as follows: initial denaturation at 95°C for 10 minutes, followed by 30 cycles at 95°C for 10 seconds, annealing at 58.5°C for 5 seconds, and extension at 72°C for 8 seconds. PCR products were quantified via molten curve at 84°C. All results were normalized to -actin to ensure a uniform amount of RNA template. Statistical analysis. The Statistical Package for Social Science (SPSS, Chicago, IL) version 17.0 was used. A nonparametric test was used to compare Kindlin-2 expression in tumor tissues and paraneoplastic tissues. Differences of Kindlin-2 expression and clinical and pathologic variables were compared using the Fisher exact test or the Cochran–Armitage trend test in the case of ordered categories. Overall survival time was calculated from the time since surgery to the last follow-up evaluation or date of death. The median follow-up time for survival was 37.1 months (range, 5–77 mo). Progression-free survival was calculated from the time of surgery to the time of first recurrence and the time from surgery to the last follow-up evaluation/death for patients who did not recur. Univariate survival analysis (overall and progression free) was estimated using Kaplan–Meier methods, and survival time was compared using the log-rank test. The Cox proportional hazards model was used for multivariate analysis. P values were considered significant at a P value of less than .05.
Results Kindlin-2 expression is high at protein and RNA levels in gastric cancer tissues We characterized the expression of Kindlin-2 in 40 paired gastric cancer and paraneoplastic tissues by Western blot. The results were measured by the ratio of Kindlin-2 and -actin gray values. We found that the mean value of Kindlin-2/-actin was higher in gastric cancer tissues than in paraneoplastic tissues (.413 ⫾ .366 and .133 ⫾ .186, respectively; P ⫽ .014; Fig. 1). High expression of Kindlin-2 (normalized tumor expression/normalized paraneoplastic expression, ⬎2) was found in 22 tumors (55% of patients), whereas relative low (normalized tumor expression/normalized paraneoplastic expression ⱕ2) expression was found in 18 tumors (45% of cases). Messenger RNA (mRNA) expression levels of Kindlin-2 was detected by real-time PCR. We found that
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Figure 1 (A) Expression of Kindlin-2 at the protein level in tumor and normal tissues. Western blot samples were made from tumor tissues of gastric cancer patients and normal paraneoplastic tissues of the same patients. Western blot gray values were normalized with -actin gray values of the same samples. The results show an increased protein level expression of Kindlin-2 in tumor tissues (the result was mean ⫾ standard deviation, n ⫽ 40). (B) Expression of Kindlin-2 at the protein level in gastric cancer tissues and paraneoplastic tissues (Western blot). T ⫽ gastric cancer tissue; N ⫽ paraneoplastic tissue.
Kindlin-2 mRNA was detectable in all corresponding paraneoplastic and gastric cancer tissues, but the levels of expression in gastric cancer tissues were much higher than corresponding paraneoplastic tissues (Kindlin-2/actin mRNA expression: 1.02 ⫾ 0.17 vs 0.75 ⫾ .19; P ⫽ .027; Fig. 2).
Kindlin-2 protein expression is related to TNM stage, invasion depth, and lymph node metastasis in gastric cancer There was no correlation between Kindlin-2 protein expression levels (high vs low) and age, sex, or tumor differentiation. There was increased Kindlin-2 protein expression in patients with poor TNM stage, deeper invasion depth, and more lymph node metastasis. Kindlin-2 protein expression levels in locally invasive T3 and T4 tumors were significantly higher when compared with those in less aggressive T1 and T2 tumors (P ⫽ .014).
Kindlin-2 expression levels in Stage II through IV tumors also were significantly higher compared with the expression levels in stages 0 and I tumors (P ⫽ .014). Kindlin-2 expression levels in patients who had more lymph node metastasis (N2, N3) were higher compared with that in patients with a fewer lymph node metastasis (P ⫽ .012; Fisher exact test; Table 2) and there was even a trend showing increasing levels of Kindlin-2 expression on tumors as the node status increased (P ⫽ .003, Cochran– Armitage trend test).
High Kindlin-2 protein expression correlates with poor overall survival and progression-free survival The correlation between Kindlin-2 protein expression levels (high/low) and patient survival with gastric cancer
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Figure 2 Expression of Kindlin-2 at the RNA level in the tumor and normal groups. RNA samples were made from tumor tissues of gastric cancer patients and normal paraneoplastic tissues of the same patients. Real-time PCR results were normalized with -actin results of the same samples. The results show an increased RNA level expression of Kindlin-2 in tumor tissues (the result was mean ⫾ standard deviation, n ⫽ 40).
after surgery was investigated with the Kaplan–Meier method (rate of follow-up evaluation is 100%: 40 of 40). Patients who had high Kindlin-2 expression levels had a significantly shorter overall survival (mean survival time, 20.0 ⫾ 3.71 mo) compared with low Kindlin-2 expression (mean survival time, 62.7 ⫾ 10.3 mo; P ⫽ .012; Fig. 3). Length of progression-free survival of patients with high Kindlin-2 expression levels also was shorter compared with patients with low Kindlin-2 expression (mean, 53.9 ⫾ 10.1 vs 11.4 ⫾ 3.52 mo for low and high Kindlin-2 expression levels, respectively; P ⫽ .012; Fig. 4).
Kindlin-2 protein expression is an independent risk factor affecting progression survival, but not overall survival Because of the correlation between Kindlin-2 expression and tumor stage, tumor invasion depth, and lymph node metastasis, therefore, only age, sex, tumor differentiation, and distant metastasis were examined by multivariate analysis for overall survival and progressionfree survival by the Cox proportional hazards model. We found that Kindlin-2 protein expression was not an independent risk factor of overall survival in gastric cancer (Table 3), but was an independent risk factor of worse progression-free survival (hazard ratio, 5.2; 95% confidence interval, 1.1–3.2; P ⫽ .032; Table 4).
Comments The novel adaptor protein Kindlin-2 is essential for integrin-mediated cell–ECM adhesions. It can interact with cytoplasmic tails of -integrin and subsequently binds to migfilin and recruits it to these focal adhesion sites.12 The presence of migfilin in focal adhesions facilitated the accumulation of other migfilin-binding proteins such as vasodilator-stimulated phosphoprotein, a regulator of actin cytoskeletal dynamics, and filamin, an actin binding protein.20 Moreover, Kindling-2 acts as a crucial co-activator in integrin activation. Ma et al21 showed that co-transfection of Kindlin-2 with talin-H results in a synergistic enhancement of integrin ␣IIb3 activation. Furthermore, short interfering RNA knockdown of endogenous Kindlin-2 impaired talin-induced ␣IIb␣3 activation in transfected Chinese hamster ovary cells and inhibited ␣v3-mediated adhesion and migration of endothelial cells. Kindlin-2 is considered a bidirectional integrin signaling protein involved in the activation of integrin adhesion to ECM and subsequently mediating integrin binding to actin cytoskeleton. Integrin functions in cell-to-cell and cell-to-ECM adhesive interactions and transduce signals from the ECM to the cell interior and vice versa (eg, mediating ECM influence on cell growth and differentiation). Because these properties implicate integrin involvement in cell migration, invasion, intravasation and extravasation, and platelet interaction, a role for integrins in tumor growth
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Correlation between Kindlin-2 and clinicopathologic factors Expression of Kindlin-2 [number (%)]
Age, y ⱕ60 ⬎60 Sex Male Female Tumor differentiation Well Moderately Poorly Pathologic stage I–II III–IV Tumor invasion depth T1–2 T3–4 Lymph node status N1 N2 N3 Distant metastasis Negative Positive
Number (n ⫽ 40)
Low expression (n ⫽ 18)
High expression (n ⫽ 22)
14 26
4 (28.6) 14 (53.8)
10 (71.4) 12 (46.2)
30 10
14 (46.7) 4 (40.0)
16 (53.3) 6 (60.0)
4 8 28
3 (75.0) 5 (62.5) 10 (35.7)
1 (25.0) 3 (37.5) 18 (64.3)
8 32
7 (87.5) 11 (34.4)
1 (12.5) 21 (65.6)
P value, Fisher exact test .186 1.000 .234
.014* .014* 8 32
7 (87.5) 11 (34.4)
1 (12.5) 21 (65.6)
21 10 9
14 (66.7) 3 (30.0) 1 (11.1)
7 (33.3) 7 (70.0) 8 (88.9)
37 3
18 (48.6) 0 (0.0)
19 (51.4) 3 (100.0)
.012*
.238
*Significant at the P ⬍ .05 level.
and metastasis is obvious. As the main link between a cell and the ECM, integrin has an essential role in the invasion process. The cellular integrin expression pattern is highly variable between cancer types. Matsuoka et al22 found that integrin ␣21 expression was associated with tumor progression and invasion in gastric cancer. Kindlin-2 as an activator of integrin might be involved in the development of cancers. It is known that dissemination and metastasis are crucial risk factors of poor prognosis in gastric cancer patients.23–26 Peritoneal dissemination is a common reason why gastric cancer cannot be resected.23 Moreover, peritoneal recurrence after curative resection was identified as a major type of recurrence (29%⬃44%).24,25 Lymph node status is an important prognostic factor regarding long-term survival of gastric cancer patients.26 The 5-year survival rate (after surgical treatment) was 86.1% in the patient group with N0 status, whereas survival rates dramatically decreased to 58.1%, 23.3%, and 5.9% in patient groups with N1, N2, and N3 status, respectively.27 Kindlin-2 has been studied in only a few cancers (eg, breast cancer17 and mesenchymal cancer).18 There are no reports on the relationship between Kindlin-2 and clinicopathologic factors of cancer or Kindlin-2 expression in gastric cancer. In our study, we found that most gastric cancer tissues (85%) had higher Kindlin-2 expression as
compared with normal tissues, and 55% of tumors had very high Kindlin-2 expression (gastric cancer tissues/normal tissues, ⱖ2). We further analyzed the relationship between Kindlin-2 protein expression and clinicopathologic factors. An interesting finding was that gastric cancer tissues with high Kindlin-2 protein expression had deeper invasion and more metastases in lymph nodes and poor TNM stage. Therefore, high Kindlin-2 protein expression might be a factor related to more aggressive biological behavior of gastric cancer. In our study, Kindlin-2 protein expression was significantly higher in stages II through IV than in stages 0 and I tumors. Kindlin-2 expression was related to the depth of tumor invasion and lymph node metastasis. There was no statistical difference in Kindlin-2 expression between patients with or without distant metastasis, which may be owing to the low number of patients3 who had distant metastasis. In addition, we found that patients with high Kindlin-2 expression level had poor overall and progression-free survival. Further, Kindlin-2 expression level was an independent risk factor in progression-free survival, but not in overall survival. High Kindlin-2 expression might facilitate integrin-mediated ECM adhesion and integrin–actin cytoskeleton binding, possibly improving the survival and proliferation of tumor cells, but this requires further research. Overall, Kindlin-2 might
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The American Journal of Surgery, Vol 203, No 2, February 2012 Table 3 Multivariate analysis of overall survival for patients with gastric cancer using the Cox model 95.0% confidence interval for HR
Kindlin-2 Sex Age Differentiation Distant metastasis
P value
HR
Lower
Upper
.175 .425 .267 .430 .248
2.832 .595 .482 1.690 4.202
.630 .166 .133 .460 .368
12.736 2.132 1.750 6.219 48.046
Kindlin-2 low expression, male sex, age ⱕ 60 years, well differentiation, and negative distant metastasis were used as reference categories. HR ⫽ hazard ratio.
Figure 3 Gastric cancer patients with high Kindlin-2 expression in tumors show poor overall survival. Kindlin-2 expression was measured by Western blot in tumors of gastric cancer patients and the patients’ survival was followed up for 89 months (Kaplan– Meier curve, log-rank, P ⫽ .012).
be a marker of poor TNM stage and prognosis of gastric cancer and might become a potential new factor used to evaluate the invasion, metastasis, and outcome of gastric cancer. Li et al28 found that the 5-year survival rate in patients with a positive expression of integrin3 mRNA was significantly lower than for those patients with a negative expression of integrin3 mRNA, and considered that integrin3 high expression can synergistically en-
hance tumor angiogenesis, and may play a crucial role in the invasion and metastasis of gastric carcinoma. However, to our knowledge, the role of Kindlin-2 expression in the prognosis of cancer patients is not clear. Further research is needed to discover the molecular biological mechanism of Kindlin-2 expression in gastric cancer invasion and metastasis affecting the outcome of patients. Moreover, most patients with gastric cancer are diagnosed in the advanced stage, surgery can be performed in only 30% of patients, and a relapse frequently occurs.29 Currently, chemotherapeutic regimens are the mainstay of treatment against advanced gastric cancer. Although oxaliplatin, docetaxel, capecitabine, and irinotecan have shown activity in recent phase III trials and increased the armamentarium of treatment options for patients with advanced gastric cancer,30 the median survival in all randomized trials remains unsatisfactory.31,32 Novel treatment options are urgently needed to improve the outcome of patients with advanced gastric cancer. Our study shows that high Kindlin-2 expression leads to poor
Table 4 Multivariate analysis of the progression-free survival for the gastric cancer with Cox model 95.0% confidence interval for HR
Kindlin-2 Sex Age Differentiation Distant metastasis Figure 4 Poor progression-free survival found in gastric cancer patients with high Kindlin-2 expression in tumors. Kindlin-2 expression was measured by Western blot in tumors of gastric cancer patients and the patients’ progression-free survival was followed up for 84 months (Kaplan–Meier curve, log-rank, P ⫽ .003).
P value
HR
Lower
Upper
.032* .679 .619 .628 .068
5.168 .752 .744 1.277 14.866
1.148 .195 .232 .476 .821
3.272 2.900 2.390 3.429 269.031
Kindlin-2 low expression, male sex, age ⱕ 60 years, well differentiation, and negative distant metastasis were used as reference categories. HR ⫽ hazard ratio. *Significant at the P ⬍ .05 level.
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TNM stage and prognosis of patients, although low Kindlin-2 expression has a better prognosis and TNM stage. Therefore, Kindlin-2 might be a candidate target for therapy to change the outcome of patients with advanced gastric cancer.
Acknowledgments The authors would like to thank Ms. Lingyi Dong and Ms. Sanna Vainionpää for excellent technical assistance with the experiment.
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