Background
Urothelial carcinoma of the upper urinary tract (UC-UUT) is relatively uncommon, accounting for <10% of all urothelial malignancies, but its incidence is increasing [
1]. Many patients who undergo curative resection develop systemic metastases within a few years, so the prognosis of this cancer is poor [
2], presumably due to occult micrometastasis at the time of surgery because of the thin walls and rich lymphatic drainage of the ureter.
Metastasis involves the spread of tumor cells from the primary tumor to a distant site [
3], and is the major cause of human cancer death. Various pathological studies have shown that poorly differentiated cancer, muscle invasion, lymph node metastasis, and lymphovascular invasion (LVI) are associated with recurrence and are unfavorable prognostic factors for UC-UUT [
2,
4,
5]. Thus, LVI and lymph node metastasis are used to predict the prognosis. Despite their clinical importance, little is known about the molecular mechanisms of LVI and lymph node metastasis, making it important to examine the factors playing a role in LVI and lymph node metastasis of UC-UUT.
Members of the Rho small GTPases family, prototype RhoA, Rac1, and Cdc42, are involved in the regulation of a variety of cellular processes, such as organization of the microfilament network, cell-cell contact, and malignant transformation, and also perform essential and specialized functions during organization of the actin cytoskeleton [
6]. RhoA regulates the formation of stress fibers and focal adhesions in cells, while Rac1 regulates the formation of lamellipodia and membrane ruffling, and Cdc42 regulates the formation of filopodia [
6,
7]. In addition, a number of investigations have established a significant role of GTPases from the Rho family in several human tumors, including UC-UUT [
7,
8]. Rac1 is ubiquitously expressed and exists in two conformational states. In response to extracellular signals, interconversion of these two states occurs via guanine nucleotide exchange factors (GEFs), which convert the inactive GDP-bound form of Rac1 to its active GTP-bound form, while GTPase-activating proteins (GAPs) inactivate proteins (GAPs) inactivate Rac1. After activation, Rac1 interacts with various specific effectors to coordinate the activation of a multitude of signaling cascades that influence diverse physiological outcomes. The Pak (p21-activated kinase) serine/threonine kinases have recently been found to be key regulators of cytoskeletal remolding, cell motility, and cell proliferation, with a role in both carcinogenesis and cellular invasion [
9]. It has been reported that Pak1, the best characterized member of this family, shows increased expression and activity in human cancers [
9‐
11]. Multiple signalling pathways converge to promote activation of Pak1 through both small GTPases and several of the tyrosine kinases. In turn, activated Pak1 regulates diverse cellular functions. Pak1 binds to Rac1 in a GTP-dependent manner, after which activated Pak1 regulates cellular functions such as cytoskeletal dynamics, cell adhesion, and transcription [
9]. Rac1 signals through Pak1 to activate c-Jun N-terminal kinase (JNK) [
9], placing Rac1 between the Ras small GTPases (Ras) and mitogen-activated protein kinase (MAPK) in the signaling cascade from growth factor receptors and v-Src to activation of JNK [
12,
13]. Gao et al. reported that a low molecular weight inhibitor of Rac GTPase targeting the activation of Rac by GEF was able to reverse the tumor cell phenotype associated with deregulation of Rac [
14]. In addition, several low molecular weight inhibitors have been shown to interfere with Pak kinase activity or function [
9]. These findings suggest that Rac1 and Pak may be potential molecular targets for the treatment of cancer.
Regarding the expression of Rho family GTPases in human cancers, most previous reports were based on the investigation of protein expression levels by Western blotting and immunohistochemistry. However, only the active GTP-binding form (active GTPase) recognizes target proteins and generates a response. Increased activity of Rac1 and overexpression of Pak1 are associated with the progression of cancer, but most of the evidence has come from cell culture studies. Therefore, the role of active Rac1 GTPase and its downstream effector needs to be studied by using surgically resected samples of human tumors to better assess their contribution to human cancer. Accordingly, we examined the expression of active GTP-bound Rac1 (Rac1 activity) and its downstream effector Pak1 in the primary tumors and metastatic lymph nodes of patients with UC-UUT by Western blotting, and also assessed the relation of these molecules with clinicopathological features. There have been few reports about the simultaneous analysis of Rac1 and Pak1 in human tumor tissues. Such information could be useful for developing individualized treatment strategies and could potentially improve the design and application of adjuvant therapy for UC-UUT.
Discussion
Rac1 and Pak1 have recently been shown to be key regulators of cancer cell signaling networks, and there are several lines of evidence linking Rac1 and Pak1 to the acquisition of migratory, invasive, and metastatic phenotypes [
7,
9]. In order to take into account possible inter-individual variations of Rac1 activity and Pak1 protein expression in UC-UUT, we performed a comparison among paired samples of tumor tissue, metastatic lymph node tissue, and non-tumor tissue from the same patient. The present study showed that Rac1 activity and Pak1 expression were significantly increased in primary tumors and metastatic lymph nodes compared with non-tumor tissues. Also, an increase of Rac1 activity and Pak1 expression in the primary tumor was correlated with poorly differentiated cancer, local invasion, lymph node metastasis, LVI, and an unfavorable prognosis. To our knowledge, this is the first report about Rac1 and Pak1 in UC-UUT. Our data suggested that Rac1 and its downstream effector Pak1 may be involved in the progression of this cancer.
As well as our findings in patients with UC-UUT, overexpression of Rac1 and Pak1 has been reported in several other human cancers [
7,
9]. Moreover, an increase of Rac1 and Pak1 activity or overexpression have been observed in breast cancer tissues and metastatic lymph nodes [
24,
25]. In the present study, increased Rac1 activity and higher Pak1 expression in the primary tumors was related to muscle invasion and lymph node metastasis. Therefore, it is likely that Rac1 and Pak1 have a role in determining the local invasive and metastatic potential of various human cancers.
Regarding the site of initial postoperative recurrence in patients who were pT
anypN0 M0 at the time of nephroureterectomy, patients with postoperative lymph node recurrence had a worse prognosis than those with bladder recurrence, probably because many bladder cancers were detected at the superficial stage by active surveillance. In the present study, LVI, Pak1 activity, Rac1, pT stage, and tumor grade were related to postoperative recurrence according to univariate analysis, with both LVI and Pak1 still being significant determinants according to multivariate analysis. As shown in Figure
7, Rac1 activity and Pak1 expression were higher in the primary tumors of patients with postoperative lymph node metastasis than in those of patients with bladder recurrence from the LVI(+) group, but not the LVI(-) group. Pak1 expression was higher in the tumors of patients with bladder recurrence than in recurrence-free patients from the LVI(+) group, but Rac1 did not differ between them. Moreover, all patients with lymph node involvement at nephroureterectomy had LVI(+) tumors on pathological examination. Therefore, LVI might be an important step along the road to lymph node metastasis. Primary tumors with LVI(+) and lymph node metastasis showed an increase of Rac1 activity and Pak1 expression, while metastatic lymph node tissues showed higher Rac1 activity and Pak1 expression than normal lymph nodes, indicating that Rac1 and Pak1 are involved in tumor metastasis. However, it is unclear whether Rac1 and Pak1 play a similar role in lymph node metastasis and bladder recurrence. It is well known that urothelial cancer often behaves like a field change disease, with multiple occurrences and recurrences due to implantation and migration, making it difficult to determine whether a recurrent tumor has been caused by tumor cell implantation, migration, or multifocal carcinogenesis [
26]. A recent molecular study revealed that UC-UUT might be less genetically stable than bladder tumors [
27]. Therefore, an increase of Rac1 activity and upregulation of Pak1 expression might play a role in lymph node metastasis of UC-UTT after nephroureterectomy, rather than contributing to bladder recurrence. The differences of molecular mechanisms between LVI and lymph node metastasis or bladder recurrence need to be investigated further. On the other hand, a previous study of bladder cancer showed that high Pak1 expression was associated with a higher risk of recurrence, even in patients with low grade/stage tumors [
28]. Taken together, therefore, it is likely that Pak1 and Rac1 both play an important role in the invasion, metastasis, and recurrence of urothelial cancer.
Although Pak1 is well known as a downstream effector of Rac1, there have been few simultaneous analyses of Rac1 and Pak1 expression in human tumor tissues. Our study showed a positive correlation between Rac1 activity and Pak1 expression in tumor tissue, while no such relation was observed in metastatic lymph node tissue. In contrast to investigation of Rac1 activity, we only measured Pak1 protein expression, but we could still determine the approximate relation between the two molecules. As shown in Figure
5, there was a positive correlation in tumor tissues, but the absolute correlation was fairly weak and no correlation was found in metastatic lymph nodes. Pak1 is the best-characterized downstream effector of Rac1, but it is also an important convergence point for many signaling pathways (including small GTPases and several tyrosine kinases) that are often activated in cancer cells [
9‐
11]. Therefore, several oncogenic pathways may act through Pak1 to promote cancer progression, so that Pak1 protein expression had a greater impact on overall and recurrence-free survival than Rac1 activity in the present study. We did not assess Pak1 activity in this study, so its activity in tumor tissues needs to be elucidated in the future.
Cell migration is central to metastasis by malignant tumors [
3]. Members of the Rho small GTPases family regulate formation of stress fibers, focal adhesions, and cell migration through reorganization of the actin cytoskeleton [
6]. Several lines of evidence have directly linked Rac1 and Pak1 to acquisition of a migratory, invasive, and metastatic phenotype and to a variety of processes that occur in tumors, including cell transformation, survival, invasion, metastasis, and angiogenesis [
7,
9]. Our findings suggested that Rac1 and Pak1 were associated with LVI and RPLN, as well as distant metastasis of UC-UUT.
The renal pelvis and ureter have thin walls and a rich lymphatic drainage [
1], so many patients present with local invasion and/or lymph node metastasis, while delay in making the initial diagnosis is correlated with a higher stage at presentation. Preoperative staging by CT/MRI was very useful for detecting patients with local invasion and/or lymph node metastasis in the present study, but the prognosis of such patients was poor (Table
1). Although systemic M-VAC therapy reduced the tumor burden of our patients with urothelial cancer, the prognosis was worse than we expected [
17]. In the present study, M-VAC therapy was performed as adjuvant chemotherapy for the 35 patients who showed lymph node or distant metastasis at surgery or during postoperative surveillance, but all of these patients died of their cancer. These 35 tumors were all LVI(+) and showed increased Rac1 activity and high Pak1 expression. Furthermore, an increase of Rac1 activity and a higher Pak1 expression were associated with a shorter overall survival time in all patients and shorter postoperative disease-free survival in pT
anypN0 M0 patients, indicating that Rac1 activity and Pak1 expression may be useful prognostic indicators for UC-UTT. These findings suggests that patients with LVI(+) tumors that have higher Rac1 activity and Pak1 expression are at more risk of developing postoperative RPLN or distant metastases in comparison to patients without these markers. Therefore, we need to assess the potential of chemotherapy for patients who have pT
anypN0 M0 tumors that are LVI(+) with increased Rac1 activity and Pak1 expression to prevent RPLN recurrence or distant metastasis by performing a randomized study in the future. In premenopausal breast cancer patients, Pak1 overexpression has been closely linked with tamoxifen resistance of tumors [
29]. On the other hand, several low molecular weight inhibitors have been shown to interfere with Pak1 kinase activity or function [
9]. Therefore, the Rac1-Pak1 pathway might be a potential therapeutic target for the prevention of tumor invasion and metastasis by inhibition of this signaling pathway. Accordingly, we should examine the effects of various inhibitors using cell lines or tumor tissue samples. Furthermore, as the Rac family has three isoforms and the Pak family has six isoforms, each isoform may play a different role. Therefore, it is necessary to investigate each of these isoforms in human cancers, and the information thus obtained may shed new light on treatment strategies for UC-UTT and other tumors.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
T.K.* and H.S. initiated the study, participated in its design and coordination, carried out the study, performed the statistical analysis, and drafted the manuscript. K.N., N.F., T.K., H.A. and T.O. carried out the study. K-I.Y. participated in the design of the study and helped to draft the manuscript. All authors read and approved the final manuscript.