Low PTEN expression and overexpression of phosphorylated AktSer473 and AktThr308 are associated with poor overall survival in upper tract urothelial carcinoma
- Authors:
- Published online on: October 9, 2020 https://doi.org/10.3892/ol.2020.12210
- Article Number: 347
-
Copyright: © Weng et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Malignant tumor cells derived from the urinary epithelium are called urothelial carcinomas (UCs), and these constitute the fourth most common cancer in the world (1). Bladder cancer (BC) is the most common type of UC, and accounts for 90–95% of the total UC cases (2). In Western countries, the prevalence of upper tract urothelial carcinomas (UTUCs) is notably less compared with that of BC, which accounts for only 5–10% of all UC cases, and the incidence of UTUC in the renal pelvis is ~2-3-fold more common than that in the ureter, and the male-to-female ratio is ~2-3:1 (3). However, in Taiwan, UTUCs account for 30% of all UC cases, and the incidence of UTUC in the renal pelvis is similar to that in the ureter with a ratio of ~1.1:0.9 (4). In addition, there are more female patients with UTUC than males (5). The high recurrence rate, high progression potential and frequent distant metastasis are the main reasons why UC has poor clinical outcomes even when diagnosed at an early stage. In a clinical setting, previous studies have shown that tumor (T) stage, tumor grade, tumor size and lymph node metastasis are important prognostic predictors for UTUC (6,7). The present study aimed to identify the biomarkers of advanced UTUC based on pathological features from human tissue samples.
PTEN is a dual protein/lipid phosphatase that dephosphorylates phosphatidyl-inositol (3,4,5)-triphosphate (PIP3) into phosphatidylinositol 4,5-biphosphate (PIP2). Deletion, mutation or silencing due to high levels of promoter methylation causes loss of PTEN activity in a number of primary and metastatic cancers (8,9). Moreover, loss of PTEN is associated with an aggressive tumor phenotype and poor clinical outcomes in UC (10). Concordantly, PTEN is also a negative regulator of the PI3K/Akt/mTOR signaling pathway (11) (Fig. 1). Akt is recruited to the cell membrane by PIP3, and Phosphoinositide-dependent kinase-1 (PDK1) phosphorylates Akt on threonine (Thr)308 (12). The mammalian target of rapamycin complex 2 (mTORC2) complex then phosphorylates Akt on serine (Ser)473 via a positive feedback loop (13), leading to full activation of Akt. Phosphorylated Akt activates mTORC1 to regulate the phosphorylation of the S6 protein and the initial translational factor in eukaryotes, eukaryotic initiation factor 4E binding protein 1 (4E-BP1) (14). Activated Akt phosphorylates several downstream effectors that regulate a variety of essential processes such as cell growth, cell metabolism, cell survival and protein synthesis (15). Although the phosphorylation of both Thr308 and Ser473 is thought to be mandatory for complete activation of the Akt pathway, there are still discrepancies about the association of this with clinical outcomes. Gallay et al (16) demonstrated that the levels of phosphorylation on Thr308, instead of on Ser473, were significantly associated with poor clinical outcomes, including overall survival, event-free survival and relapse-free survival, in acute myeloid leukemia. By contrast, Freudlsperger et al (17) showed that the relative levels of phosphorylated (p)Akt on Ser473 were associated with overall survival and progression-free survival in patients with advanced head and neck squamous cell carcinoma, but the relative levels of pAkt on Thr308 were not correlated with patient outcomes.
Activation of PI3K and Akt is reported to induce ovarian (18), breast (18,19), esophageal (20) and pancreatic cancer (21), among other (9,22). The PI3K/Akt/mTOR signaling pathway serves an essential role in various cellular processes, including cell growth, cell motility, cell survival, angiogenesis and cell metabolism (23–25). This pathway is also documented to be associated with carcinogenesis in UC (26), and plays a central role in resistance to chemotherapy and radiation therapy in multiple cancer types (27).
In the present study, PTEN gene alterations were investigated using fluorescence in-situ hybridization (FISH), and the protein expression levels of PTEN, phosphorylation of Ser473 in Akt (pAktSer473) and phosphorylation of Thr308 in Akt (pAktThr308) were analyzed using immunohistochemistry (IHC) in UTUC tissues from 65 patients.
Materials and methods
Patients and tissue samples
Patients with confirmed diagnosis of UTUC after ureteroscopic biopsy or computed tomography-guided biopsy, who then underwent nephroureterectomy between January 2007 and October 2014, were included in the present study. Patients with insufficient tissue for complete pathologic review, including the diagnosis of atypical urothelial cells, or without the required clinical data, including demographic and survival information, were excluded. An informed consent form regarding the utilization of residual tissues for medical research was signed at the outpatient visit after explanation by the physician, and all specimens were sent to the tissue bank of Linkou Chang Gung Memorial Hospital (Taoyuan, Taiwan) immediately after the operation at 4°C and then stored at −80°C. The present study was approved by The Human Subject Research Ethics Committee/Institutional Review Board (IRB) of Linkou Chang Gung Memorial Hospital (approval no. 201601555B0). The clinicopathological details of 65 patients with UTUC were analyzed in the study. In total, 30 (46%) patients were male and 35 (54%) were female, with a median age of 77 years (range, 49–98 years). Additionally, 22 slides of normal kidney (19 slides) and ureter (3 slides) tissues were obtained to use as reference. According to guidelines (28,29), nephroureterectomy would be the standard treatment for these patients. As for T3 or T4 lesions, neoadjuvant chemotherapy would be applied if patients were fit enough, followed by surgery if resectable. Adjuvant systemic treatment was initiated if recurrence was found. For all patients, clinical data, including age, sex, history of smoking, alcohol consumption, diabetes mellitus, hypertension, tumor location, histology grade, tumor stage based on the American Joint Committee on Cancer classification (30), metastasis, recurrence and urolithiasis, were recorded (Table I). The overall survival was calculated independently and stratified according to the noteworthy parameters.
FISH for analysis of the PTEN gene
Two commercially available dual-color FISH probes (CytoTest Inc.; cat no. CT-PAC101 and CT-LSP042) were designed to detect copy number changes in the region of the human PTEN gene, which is located on chromosome 10q23. The probes hybridized to chromosome 10 in both metaphase and interphase and the Locus Specific Probe (LSP), which is around 470 kb in length, exhibited an orange fluorescent signal under the appropriate filters. The other probe, the Chromosome 10 Counting Probe (CCP10), exhibiting a green signal, served as an internal control according to the nature derived from chromosome 10-specific pericentromeric DNA. First, 4-µm-thick formalin-fixed paraffin-embedded (FFPE) samples were deparaffinized in 3 washes of xylene for 5 min each, and then samples were rehydrated using a descending ethanol series (100, 85 and 70%). Samples were washed in 4X saline sodium citrate (SSC) at room temperature (RT) for 30 min in a rotating shaker, and then were treated with 1 M sodium thiocyanate (NaSCN) at RT overnight. Lastly, slides were washed in distilled water for 5 min. The specimens were digested in 250 µm 10% pepsin in 0.01 M HCl. Probes and target DNA were then co-denatured at 82°C for 10 min and hybridized overnight in a 37°C incubator. Post-hybridization washes were performed in 2X SSC at RT for 5 min and in 0.3% NP40/2X SSC at 73°C for 2 min, followed by a 1-min wash at RT in distilled water. All images were captured using a Leica DM2500 fluorescence microscope (Leica Microsystems GmbH) using a magnification of ×63 with an ASI CCD camera (CCD-1300DS; Applied Spectral Imaging), and were subsequently analyzed with FISHView EXPO version 5.5 software (Applied Spectral Imaging). To evaluate the PTEN copy number, signals in 300 non-overlapping nuclei in each sample were counted using the aforementioned software. DAPI staining of nuclei (using 1 µg/ml at room temperature for 30 min) was performed after resuspension of the cell pellet into absolute ethanol at −20°C (Sigma-Aldrich; Merck KGaA). After staining, the slides were ready for interpretation in reference to the corresponding hematoxylin and eosin (H&E)-stained tissue identified in the areas of carcinoma. H&E staining was performed by the hospital according to routine protocols and a series of 10 slides for the same patient was retrieved after IRB approval. Heterozygous deletion of PTEN was defined as a ratio of PTEN signal/centromere 10 probe signal <0.5. In addition, homozygous deletion of PTEN was defined as a complete absence of PTEN probe signal in >60% of tumor nuclei per sample, but there could be 1–2 PTEN signals in adjacent cells.
IHC for PTEN, pAktSer473 and pAktThr308
A total of 65 FFPE UTUC tissue samples were collected between January 2007 and October 2014. The areas of carcinoma were identified by three researchers, including one pathologist, in H&E-stained tissues. IHC staining was performed in the present study using primary antibodies against PTEN (clone D4.3 XP; 1:250 dilution; cat. no. 9188), pAktSer473 (clone 736E11; 1:200 dilution; cat. no. 3787) and pAktThr308 (clone 244F9; 1:100 dilution; cat. no. 4056) (all Cell Signaling Technology, Inc.). All 4-µm FFPE UTUC tissues were deparaffinized with xylene and rehydrated in 100, 85 and 70% ethanol (Sigma-Aldrich; Merck KGaA). The procedure was performed according to that reported in our previous study with modifications (31). Antigen retrieval was performed by heating the slides at 95°C in sodium citrate buffer (10 mM sodium citrate, 0.05% Tween-20, pH 6.0) for 20 min. Endogenous peroxidase activity was quenched by incubation with hydrogen peroxidase (Thermo Fisher Scientific, Inc.) for 10 min at RT, followed by an ultraviolet block (Thermo Fisher Scientific, Inc.) for 5 min at RT to prevent non-specific background staining. Slides were incubated for 16 h at 4°C with the aforementioned rabbit anti-human PTEN, pAKTSer473 and pAKTThr308 antibodies for each group. After 16 h of incubation, the slides were left for 1 h at RT, followed by primary antibody amplifier Quanto (Thermo Fisher Scientific, Inc.) treatment for 10 min at RT. After application of the secondary antibody for 10 min at RT (UltraVision Quanto Detection System; cat. no. TL-060-QHD; ready to use; Thermo Fisher Scientific, Inc.), the slides were stained using the chromogen 3,3′-diaminobenzidine tetrahydrochloride (Dako; Agilent Technologies, Inc.) for 20 sec at RT. Finally, all slides were counterstained with hematoxylin for 20 sec, dehydrated in 95 and 99% ethanol for 2 min each, and mounted, all at RT. Negative control slides were incubated with PBS at 4°C overnight. Slides from normal kidney or ureter were stained as aforementioned and used as positive controls. All slides were scanned using a high-resolution brightfield APERIO® ScanScope (Leica Microsystems, Inc.) at ×40 magnification, and digital images were used for scoring of the immunoreactivity of targeted proteins.
Scoring of PTEN, pAktSer473 and pAktThr308 protein expression levels
The IHC score in each case was independently evaluated by one pathologist, one doctor and one researcher. Tumors stained by each marker were evaluated for the location of staining (nuclear or cytoplasmic), extent of staining (percentage of positive cells, 0–100%) and intensity of staining (0, negative; 1, weak; 2, medium; and 3, intense), and a cut-off value was based on the median H-score in tumor (32). Adjacent normal tissues were defined as areas surrounding the tumor in each section with confirmation by pathologist in case of uncertainty. To determine the percentage of positive cells and the staining intensity, the H-score was calculated by the sum of the products of the intensity and extent of expression scores, obtaining a value from 0 to 300. For statistical analysis, the final H-score was divided into two scoring categories. First, cases with H-scores ≥ the median were considered to have high expression; by contrast, cases with H-scores < the median were considered to have low expression (Table II). Second, the results for the nuclear to cytoplasmic expression ratio were stratified into four groups: Nuclear high/cytoplasmic high, nuclear high/cytoplasmic low, nuclear low/cytoplasmic high and nuclear low/cytoplasmic low.
 | Table II.Immunohistochemistry distribution in adjacent normal vs. tumor tissues, and association between cytoplasmic and nuclear expression levels. |
Statistical analysis
The expression levels of protein in adjacent normal tissues and UTUCs were compared using non-parametric statistics. The association between PTEN gene alternation, protein expression levels and various clinical characteristics, including that between protein expression in UTUCs and pT stage status (low vs. high), were evaluated using χ2 test or Fisher's exact test (Table III). Two-way ANOVA with Bonferroni's correction was used to compare the differences between normal and tumor cells in the cytoplasm and nucleus, respectively. Patients who were lost to follow-up were censored on the date of the last visit. All significant parameters from the univariate analyses were included in the multivariate analyses. The probabilities of overall survival were calculated using Kaplan-Meier analysis, and log-rank tests were used to compare overall survival between patient groups. P<0.05 was considered to indicate a statistically significant difference. All statistical analyses were performed using SPSS software version 20.0 (IBM Corp.) or GraphPad Prism version 7.00 (GraphPad Software, Inc.).
Results
PTEN alterations identified using FISH
In total, 60 (92%) tumors were located in the renal pelvis and 4 (6%) tumors were located in the ureter, with 1 (2%) tumor located in both the renal pelvis and the ureter (Table I). A total of 16 (25%) patients presented with Ta, 12 (18%) patients presented with pT1, 11 (17%) patients presented with pT2, 20 (31%) patients presented with pT3 and 6 (9%) patients presented with pT4. In total, 11 (17%) patients had low-grade tumors, while the remaining 54 (83%) patients had high-grade tumors. A total of 16 (25%) patients had metastasis, and the median follow-up time was 96 months (range, 30–159 months; Table I). PTEN gene deletions were found in 33.8% (22/65) of all UTCUs (Table SI). Representative images of the FISH results are illustrated in Fig. S1. Among the specimens with PTEN deletion, 18 (81.8%) samples showed heterozygous deletion and 1 sample had a homozygous deletion. Monosomy of chromosome 10 was detected in 3 samples (data not shown). PTEN gene translocation was found in 1 of the heterozygous deletion samples. However, there was no significant association between PTEN gene alteration, protein expression (Table SI) or clinicopathological parameters (data not shown).
Protein expression levels identified using IHC
Representative patterns of protein expression levels detected by IHC are depicted in Fig. 2, and the results with further subgroup categorization based on the median H score are summarized in Table II. The distribution of protein expression levels between tumor and normal tissues was not significantly different according to the classification of expression levels above or below the median H score, with the exception of cytoplasmic spAktSer473 protein expression levels, which were lower in tumor tissues compared with those in normal tissues (P=0.003). As for PTEN expression in the nucleus, 57% of tumors and 50% of normal tissues had low expression levels (χ2=0.319; P=0.572). Similarly, for PTEN expression in the cytoplasm, 55% of tumors vs. 45% of normal tissues had low expression levels (χ2=0.651; P=0.420). As for pAktSer473 expression in the nucleus, 66% of tumors and 45% of normal tissues had low expression levels (χ2=5.241; P=0.072), while a significant difference was found in the cytoplasm with 77% of tumors vs. 41% of normal tissues having low expression (χ2=11.343; P=0.003). As for pAktThr308 expression in the nucleus, 65% of tumors and 45% of normal tissues had low expression levels (χ2=2.510; P=0.113), and a similar trend was observed in the cytoplasm with 63% of tumors vs. 45% of normal tissues having low expression (χ2=2.104; P=0.147). On the other hand, statistically significant differences were observed in the protein expression levels between tumor and adjacent normal tissues (Fig. 3). PTEN, pAktSer473 and pAktThr308 levels were all significantly lower in UTUC tissues compared with those in adjacent normal tissues in terms of H scores. The mean PTEN expression in the cytoplasm between normal and tumor tissues was 104 and 49.53, while that in the nucleus was 109.2 and 43.86, respectively; mean pAktSer473 expression in the cytoplasm between normal and tumor tissues was 68.2 and 26.9, while that in the nucleus was 77.12 and 31.86, respectively; mean pAktThr308 expression in the cytoplasm between normal and tumor tissues was 147.3 and 47.03, while that in the nucleus was 131.1 and 46.95, respectively.
Association of PTEN results identified using FISH and IHC
A total of 11/18 samples with heterozygous PTEN gene deletion showed lower nuclear and cytoplasmic PTEN protein expression compared with the expression levels of the other samples, and 7 samples had high expression levels. Overall, 2 out of 3 samples with monosomic PTEN gene deletion showed lower nuclear and cytoplasmic PTEN protein expression compared with that in the other sample, which had high expression levels. One sample with homozygous PTEN gene deletion showed no PTEN protein expression in neither the nucleus or the cytoplasm. There was no significant association between PTEN gene alteration and nuclear PTEN protein expression (P=0.434), cytoplasmic PTEN protein expression (P=0.338), nuclear pAktSer473 protein expression (P=0.423), cytoplasmic pAktSer473 protein expression (P=0.566), nuclear pAktThr308 protein expression (P=0.906) or cytoplasmic pAktThr308 protein expression (P=0.634) (Table SI).
Association of patient characteristics with protein expression levels
Table III summarizes the association of protein expression levels with clinicopathological parameters. Lower cytoplasmic PTEN protein expression levels were significantly associated with advanced T stage (T2-pT4 vs. pTa and pT1, 38 vs. 17%, P=0.023), and expression of nuclear and cytoplasmic pAktThr308 protein was associated with higher tumor grade (high grade vs. low grade, 60 vs. 5%, P=0.026 and 58 vs. 5%, P=0.031, respectively). However, pAktSer473 protein expression was not associated with T stage or tumor grade.
Univariate and multivariate analyses of clinical characteristics and protein expression levels
In the univariate analysis, high T stage [P=0.01; hazard ratio (HR)=3.40; 95% CI, 1.34–8.60), metastatic status (P=0.003; HR=3.55; 95% CI, 1.55–8.11), low cytoplasmic PTEN protein expression (P=0.016; HR=3.14, 95% CI, 1.24–7.95) and high cytoplasmic pAktSer473 protein expression (P=0.019; HR=2.71; 95% CI, 1.18–6.21) were predictive of poor overall survival. However, in the multivariate analysis, only metastatic status (P=0.031, HR=2.73, 95% CI, 1.10–6.78), low cytoplasmic PTEN protein expression (P=0.017; HR=3.29; 95% CI, 1.24–8.73) and high cytoplasmic pAktSer473 protein expression (P=0.027; HR=2.64; 95% CI, 1.12–6.23) were significantly associated with poor prognosis (Table IV).
Stratification of survival differences according to protein expression subgroups
The results of Kaplan-Meier analysis and log-rank test for overall survival according to clinicopathological parameters and protein expression are presented in Figs. 4 and 5. Survival was poor in patients with high T stage (P=0.006) (Fig. 4A), metastatic disease (P=0.001) (Fig. 4B), low cytoplasmic PTEN protein expression (P=0.011) (Fig. 4C) and high cytoplasmic pAktSer473 protein expression (P=0.014) (Fig. 4D). The co-expression phenotypes with low cytoplasmic PTEN and high cytoplasmic pAktSer473 (PTENlow/pAktSer473/high) (P<0.001) (Fig. 5A), low nuclear and/or cytoplasmic PTEN and high pAktThr308 (PTENlow/pAktThr308/high) (P=0.024) (Fig. 5B), low nuclear and/or cytoplasmic PTEN and high pAktSer473 (PTENlow/pAktSer473/high) (P=0.016) (Fig. 5C) and low cytoplasmic PTEN with high pAktSer473 and pAktThr308 (PTENlow/pAktSer473/high/pAktThr308/high) (P=0.001) (Fig. 5D) were demonstrated to have unfavorable impacts on overall survival.
Discussion
In the present study, lower protein expression levels of PTEN, pAktSer473 and pAktThr308 in either the cytoplasm or nucleus were observed in UTUC compared with those in adjacent normal tissues (P<0.001; Fig. 3). In addition, high T stage, metastatic disease, low cytoplasmic PTEN protein expression levels, high cytoplasmic pAktSer473 protein expression levels, co-expression of low cytoplasmic/nuclear PTEN and high cytoplasmic pAktThr308 and high pAktSer473 had unfavorable impacts on overall survival (Figs. 4 and 5).
Based on the results from the paired samples analyzed using IHC, the PTEN protein expression levels in either the cytoplasm or nucleus were significantly higher in adjacent normal tissues compared with those in tumor tissues (all P<0.001; Fig. 3). Therefore, alterations in gene structure were further investigated, and the majority of cases presented PTEN gene heterozygous deletions, with only one case showing a homozygous deletion, which resulted in the absence of PTEN protein expression.
Nevertheless, a previous study demonstrated that PTEN deletion in bladder cancer was significantly associated with recurrence in the Ta stage of disease and with progression in the T1 stage of disease (10). The present study focused on UTUC, and a high proportion of PTEN gene deletions (22/65; 33.8%) were observed in UTUC tissues. A previous report by Rieken et al (33) revealed that loss of PTEN protein expression was rare, and was associated with an aggressive phenotype, high tumor grade, high tumor stage and metastasis in UTUC. Further association with poor overall mortality was also documented. Although the lower number of cases showing loss of PTEN protein expression in Western countries contrasts with the higher rate in Taiwan (34), the results regarding patient outcomes were similar in the present study.
In addition, the correlation between the location of proteins in tumor cells and patient survival rate was further analyzed. Notably, it was found that patients with low cytoplasmic PTEN and high pAktSer473 expression levels (the PTENlow/pAktSer473/high group) had significantly shorter overall survival compared with that of other groups (P<0.001). Similarly, patients with low nuclear and/or cytoplasmic PTEN and high pAktThr308 expression levels (the PTENlow/pAktThr308/high group) had significantly shorter overall survival compared with that of other groups (P=0.024). Moreover, patients with low nuclear and/or cytoplasmic PTEN and high pAktSer473 expression levels (the PTENlow/pAktSer473/high group) also showed significantly shorter overall survival compared with that of other groups (P=0.016). Patients with low cytoplasmic PTEN and high pAktSer473 and pAktThr308 expression levels (the PTENlow/pAktSer473/high/pAktThr308/high group) had significantly shorter survival compared with that of other groups (P=0.001) (Fig. 5). When patients lost PTEN expression, regardless of location in the cytoplasm or nucleus, this was significantly associated with less favorable survival outcomes. Additionally, to the best of our knowledge, homozygous deletion of PTEN is uncommon in urothelial cancer (10), and the present study is the first to report this.
Contrary to expectations, the protein expression levels of pAktSer473 and pAktThr308 were lower in tumor samples compared with those in normal tissue samples (Fig. 3). These results coincide with those of a study by Munari et al (35), in which 99 archival FFPE tissues were evaluated for the expression status and prognostic significance of members of the mTOR signaling pathway in UTUC. Significantly higher expression levels of PTEN and pAkt were found in benign urothelium tissues compared with those in paired tumor samples. A possible reason for this could be that the prevalence of PTEN loss in tumors varies between 8 and 36% of cases (33), and activation of Akt phosphorylation sites is regulated by different downstream pathways.
PTEN is a tumor suppressor gene, and dephosphorylation of PIP3 into PIP2 can prevent hyperactivation of Akt (11). According to the findings of Makboul et al (36), decreased PTEN protein expression levels were significantly associated with high-grade tumors in UC and with poorly differentiated squamous cell carcinomas. A number of studies have indicated that PTEN deletion was more prevalent in the nucleus compared with that in the cytoplasm (37,38). Loss of cytoplasmic PTEN expression was primarily observed in the pT2-pT4 stages of UC, which was documented in 77% (10/13) of UC cases (39). In addition, several studies demonstrated that the active Akt protein is an important regulatory factor in cancer cells, giving rise to uncontrolled proliferation without apoptosis (26,40). For example, the activation of Akt is primarily driven by the phosphorylation of two residues, Thr308 and Ser473, which are located in the activation loop and in the C-terminal hydrophobic motif of the protein, respectively (41). The site of Ser473 phosphorylation is known to be associated with tumor formation, and its phosphorylation may be triggered by mTORC2 activation (17,42). However, protein activation mediated by phosphorylated Thr308, which in turn is regulated by PDK1, is considered necessary and sufficient to stimulate Akt signaling in cells; therefore, phosphomimetics are commonly used to study the biology of Akt signaling, although they may be insufficient in clarifying the mechanism of the whole Akt signaling pathway. Gallay et al indicated that pAktrThr308 could be a diagnostic marker of Akt activity (16). Moreover, Akt phosphorylation at Thr308 is associated with human non-small cell lung cancer (43) (Fig. 1).
In the present study, both cytoplasmic and nuclear PTEN levels in tumors were compared with those in adjacent normal cells (P<0.001; Fig. 3), and lower expression levels of cytoplasmic PTEN were found in muscle invasive disease (high stage, pT2-4) compared with those in non-muscle invasive disease (low stage, pTa-1; P=0.023; Table II). Furthermore, high expression levels of cytoplasmic and nuclear pAktThr308 were associated with high tumor grade (P=0.031 and P=0.026, respectively). Similar results from stage I lung adenocarcinoma also revealed that a PTEN(−)/pAkt(+)/pmTOR(+) phenotype was associated with poor overall survival (44). In agreement with these data, the present study showed that low cytoplasmic PTEN protein expression levels were observed in high T stage tissues, and high nuclear or cytoplasmic pAktThr308 protein expression levels were observed in high tumor grade tissues. Furthermore, it was revealed that high T stage, metastasis, low cytoplasmic PTEN protein expression and high cytoplasmic pAktSer473 protein expression levels were strong predictors of poor survival in univariate analysis (Table IV). In multivariate analysis, metastasis, low cytoplasmic PTEN protein expression levels and high cytoplasmic pAktSer473 protein expression levels remained strong predictors. Table II showed the association between clinical features and target protein levels by IHC, which showed no specific association between pAktSer473, pAktThr308 and metastasis. However, patients with high cytoplasmic/nuclear pAktThr308 possessed high tumor grades in the present cohort. Therefore, it was hypothesized that pAktSer473 and pAktThr308 are not directly associated with metastasis. It is also noteworthy that high T stage in the present cohort was not a significant predictor in the multivariate analysis. A possible reason for this phenomenon may be that cisplatin-based chemotherapy works well for patients who progress to locally advanced or metastatic disease as standard first-line systemic treatment. Therefore, in the present cohort with the majority of patients at the Ta-T2 (60%) stages, T stage is not a significant predictor for overall survival in the multivariate analysis. Cha et al, Margulis et al and Ehdaie et al (45–47) reported that UTUC with lymph node metastasis was associated with worse cancer-specific and overall survival compared with UTUC without lymph node metastasis. In addition, two other studies have shown that nuclear pAkt expression levels and pathological stage were associated with poor prognosis in UTUC (48,49), supporting the present findings and suggesting that the PTEN/PI3K/pAkt signaling pathway is important in the carcinogenesis of UTUC.
The incidence of UTUC accounts for 5–10% of that of all UCs in Western countries, while the incidence is as high as 30% in Taiwan, and UTUC is associated with consumption of Chinese herbal medicines containing aristolochic acid (AA) (50). In the present study of 65 patients with UTUC, high T stage, metastasis, low cytoplasmic PTEN protein expression levels, high cytoplasmic pAktSer473 protein expression levels and co-expression of low cytoplasmic PTEN and high pAktSer473 were strong predictors of poor overall survival. Munari et al (35) and Izquierdo et al (48) also reported that high T stage, lymph node metastasis and lymphovascular invasion were significant predictors of tumor progression and increased cancer-specific mortality in UTUC. Moreover, Shin et al (44) demonstrated that a PTEN(−)/pAkt(+)/pmTOR(+) result from IHC analysis was associated with poor prognosis in stage I non-small cell lung carcinoma. Although these aforementioned factors are highly associated with cancer prognosis when considered together, they are not synergistic and do not have any effect on their own. Koletsas et al (39) found that PTEN expression loss was not associated with Akt activation, suggesting that more complicated pathways could be involved through crosstalk or synergistic effects rather than direct activation (38). By contrast, a study evaluating the mTOR pathway members in patients with UTUC showed that none of the existing biomarkers were useful for predicting tumor progression or cancer-specific mortality (35). These findings implied that differences in ethnicity or environmental factors, such as AA, may make the clinical presentation of the disease variable and unpredictable.
There are some limitations in the present study. First, a couple of clinical factors were also associated with oncological outcomes, for example, lymph node status. However, lymph node dissection is not the routine procedure during nephroureterectomy, especially when the pre-operative image study is negative for lymph node disease in patients with UTUC. Although there were 6 patients (9%) with T4 disease, representing the high-risk group for nodal disease, the present study only incorporated distant metastasis as a predictor of long-term oncological outcome instead of addressing the association with lymph node status. Future studies should include lymph node dissection in the prospective setting. Second, FISH and IHC were performed to quantify the expression levels of targeted proteins; despite meticulous quality control and standardization of every procedure, experimental errors may still have occurred in these tests. Alternative analyses should be performed in future work, including western blotting and ELISA for determination of protein expression levels, in order to investigate associations with other notable targets. If further studies showed a significant association with the mechanism of invasion or proliferation, the levels of target proteins before and after systemic treatment could also be explored. The present study showed the preliminary results of the hypothesis, indicating that low PTEN expression with upregulation of phosphorylated AktSer473 and AktThr308 may be associated with poor overall survival in UTUC and may be applied as predictors for outcome stratification before surgery. However, in real world practice, it is hard to obtain a large enough sample size of patients with UTUC before surgery for tissue sampling. If technical advancements reach a level of improved accuracy in regard to small tissue volumes, it may be helpful to predict the outcome of surgery or to enable more personalized planning prior to surgery for patients with UTUC with positive markers.
To the best of our knowledge, the present study is the first to evaluate the association of PTEN gene alteration and the co-expression of PTEN, pAktSer473 and pAktThr308 with clinicopathological parameters and outcomes in UTUC. The present results suggest that patients with negative protein expression patterns should receive further personalized follow-up and adjuvant treatment.
Supplementary Material
Supporting Data
Acknowledgements
The authors would like to thank Ms. Shu-Ting Gan, the analyst at the Center for Big Data Analytics and Statistics (CLRPG3D0046) at the Linkou Chang Gung Memorial Hospital (Taoyuan, Taiwan) for the statistical consultation and manuscript revision.
Funding
The present study was supported by Linkou Chang Gung Memorial Hospital (grant nos. CORPG3G0081 and NTUT-CGMH-106-08).
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Authors' contributions
WHW, LCL and STP conceived and designed the study. KJY, LCL, YJP, CKC and YTC analyzed and interpreted the data. WHW, KJY and LCL drafted the initial manuscript, and YJP, STP and CKC critically revised the manuscript for important intellectual content. All authors read and approved the final manuscript.
Ethics approval and consent to participate
This study was approved by The Human Subject Research Ethics Committee/Institutional Review Board (approval no. 201601555B0) of Linkou Chang Gung Memorial Hospital (Taoyuan, Taiwan). All patients provided written informed consent.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Glossary
Abbreviations
Abbreviations:
|
UCs |
urothelial carcinomas |
|
UTUC |
upper tract urothelial carcinoma |
|
BC |
bladder cancer |
|
PIP3 | |
|
PIP2 |
phosphatidylinositol 4,5-biphosphate |
|
FFPE |
formalin-fixed, paraffin-embedded |
|
FISH |
fluorescence in situ hybridization |
References
|
Ploeg M, Aben KK and Kiemeney LA: The present and future burden of urinary bladder cancer in the world. World J Urol. 27:289–293. 2009. View Article : Google Scholar : PubMed/NCBI | |
|
Siegel R, Naishadham D and Jemal A: Cancer statistics, 2013. CA Cancer J Clin. 63:11–30. 2013. View Article : Google Scholar : PubMed/NCBI | |
|
Verhoest G, Shariat SF, Chromecki TF, Raman JD, Margulis V, Novara G, Seitz C, Remzi M, Rouprêt M, Scherr DS and Bensalah K: Predictive factors of recurrence and survival of upper tract urothelial carcinomas. World J Urol. 29:495–501. 2011. View Article : Google Scholar : PubMed/NCBI | |
|
Lai MN, Wang SM, Chen PC, Chen YY and Wang JD: Population-based case-control study of Chinese herbal products containing aristolochic acid and urinary tract cancer risk. J Natl Cancer Inst. 102:179–186. 2010. View Article : Google Scholar : PubMed/NCBI | |
|
Taiwan Health Promotion Administration Ministry of Health and Welfare Taiwan, . Cancer Registry Annual Report, 2014 Taiwan. 1212016. | |
|
Rouprêt M, Babjuk M, Compérat E, Zigeuner R, Sylvester RJ, Burger M, Cowan NC, Böhle A, Van Rhijn BW, Kaasinen E, et al: European association of urology guidelines on upper urinary tract urothelial cell carcinoma: 2015 Update. Eur Urol. 68:868–879. 2015. View Article : Google Scholar : PubMed/NCBI | |
|
Kikuchi E, Margulis V, Karakiewicz PI, Roscigno M, Mikami S, Lotan Y, Remzi M, Bolenz C, Langner C, Weizer A, et al: Lymphovascular invasion predicts clinical outcomes in patients with node-negative upper tract urothelial carcinoma. J Clin Oncol. 27:612–618. 2009. View Article : Google Scholar : PubMed/NCBI | |
|
Vivanco I and Sawyers CL: The phosphatidylinositol 3-Kinase AKT pathway in human cancer. Nat Rev Cancer. 2:489–501. 2002. View Article : Google Scholar : PubMed/NCBI | |
|
Parsons DW, Wang TL, Samuels Y, Bardelli A, Cummins JM, DeLong L, Silliman N, Ptak J, Szabo S, Willson JK, et al: Colorectal cancer: Mutations in a signalling pathway. Nature. 436:7922005. View Article : Google Scholar : PubMed/NCBI | |
|
Cordes I, Kluth M, Zygis D, Rink M, Chun F, Eichelberg C, Dahlem R, Fisch M, Höppner W, Wagner W, et al: PTEN deletions are related to disease progression and unfavourable prognosis in early bladder cancer. Histopathology. 63:670–677. 2013.PubMed/NCBI | |
|
Chalhoub N and Baker SJ: PTEN and the PI3-kinase pathway in cancer. Annu Rev Pathol. 4:127–150. 2009. View Article : Google Scholar : PubMed/NCBI | |
|
Stokoe D, Stephens LR, Copeland T, Gaffney PR, Reese CB, Painter GF, Holmes AB, McCormick F and Hawkins PT: Dual role of phosphatidylinositol-3,4,5-trisphosphate in the activation of protein kinase B. Science. 277:567–570. 1997. View Article : Google Scholar : PubMed/NCBI | |
|
Sarbassov DD, Guertin DA, Ali SM and Sabatini DM: Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science. 307:1098–1101. 2005. View Article : Google Scholar : PubMed/NCBI | |
|
Sabatini DM: mTOR and cancer: Insights into a complex relationship. Nat Rev Cancer. 6:729–734. 2006. View Article : Google Scholar : PubMed/NCBI | |
|
Hay N: The Akt-mTOR tango and its relevance to cancer. Cancer Cell. 8:179–183. 2005. View Article : Google Scholar : PubMed/NCBI | |
|
Gallay N, Dos Santos C, Cuzin L, Bousquet M, Simmonet Gouy V, Chaussade C, Attal M, Payrastre B, Demur C and Récher C: The level of AKT phosphorylation on threonine 308 but not on serine 473 is associated with high-risk cytogenetics and predicts poor overall survival in acute myeloid leukaemia. Leukemia. 23:1029–1038. 2009. View Article : Google Scholar : PubMed/NCBI | |
|
Freudlsperger C, Horn D, Weißfuß S, Weichert W, Weber KJ, Saure D, Sharma S, Dyckhoff G, Grabe N, Plinkert P, et al: Phosphorylation of AKT(Ser473) serves as an independent prognostic marker for radiosensitivity in advanced head and neck squamous cell carcinoma. Int J Cancer. 136:2775–2785. 2015. View Article : Google Scholar : PubMed/NCBI | |
|
Campbell IG, Russell SE, Choong DY, Montgomery KG, Ciavarella ML, Hooi CS, Cristiano BE, Pearson RB and Phillips WA: Mutation of the PIK3CA gene in ovarian and breast cancer. Cancer Res. 64:7678–7681. 2004. View Article : Google Scholar : PubMed/NCBI | |
|
Kirkegaard T, Witton CJ, McGlynn LM, Tovey SM, Dunne B, Lyon A and Bartlett JM: AKT activation predicts outcome in breast cancer patients treated with tamoxifen. J Pathol. 207:139–146. 2005. View Article : Google Scholar : PubMed/NCBI | |
|
Okano J, Snyder L and Rustgi AK: Genetic alterations in esophageal cancer. Methods Mol Biol. 222:131–145. 2003.PubMed/NCBI | |
|
Asano T, Yao Y, Zhu J, Li D, Abbruzzese JL and Reddy SA: The PI 3-kinase/Akt signaling pathway is activated due to aberrant Pten expression and targets transcription factors NF-kappaB and c-Myc in pancreatic cancer cells. Oncogene. 23:8571–8580. 2004. View Article : Google Scholar : PubMed/NCBI | |
|
Broderick DK, Di C, Parrett TJ, Samuels YR, Cummins JM, McLendon RE, Fults DW, Velculescu VE, Bigner DD and Yan H: Mutations of PIK3CA in anaplastic oligodendrogliomas, high-grade astrocytomas, and medulloblastomas. Cancer Res. 64:5048–5050. 2004. View Article : Google Scholar : PubMed/NCBI | |
|
Shaw RJ and Cantley LC: Ras, PI(3)K and mTOR signalling controls tumour cell growth. Nature. 441:424–430. 2006. View Article : Google Scholar : PubMed/NCBI | |
|
Knowles MA, Platt FM, Ross RL and Hurst CD: Phosphatidylinositol 3-kinase (PI3K) pathway activation in bladder cancer. Cancer Metastasis Rev. 28:305–316. 2009. View Article : Google Scholar : PubMed/NCBI | |
|
Courtney KD, Corcoran RB and Engelman JA: The PI3K pathway as drug target in human cancer. J Clin Oncol. 28:1075–1083. 2010. View Article : Google Scholar : PubMed/NCBI | |
|
Askham JM, Platt F, Chambers PA, Snowden H, Taylor CF and Knowles MA: AKT1 mutations in bladder cancer: Identification of a novel oncogenic mutation that can co-operate with E17K. Oncogene. 29:150–155. 2010. View Article : Google Scholar : PubMed/NCBI | |
|
Brognard J, Clark AS, Ni Y and Dennis PA: Akt/protein kinase B is constitutively active in non-small cell lung cancer cells and promotes cellular survival and resistance to chemotherapy and radiation. Cancer Res. 61:3986–3997. 2001.PubMed/NCBI | |
|
Clark PE, Agarwal N, Biagioli MC, Eisenberger MA, Greenberg RE, Herr HW, Inman BA, Kuban DA, Kuzel TM, Lele SM, et al: Bladder cancer. J Natl Compr Canc Netw. 11:446–475. 2013. View Article : Google Scholar : PubMed/NCBI | |
|
Rouprêt M, Babjuk M, Compérat E, Zigeuner R, Sylvester R, Burger M, Cowan N, Böhle A, Van Rhijn BW, Kaasinen E, et al: European guidelines on upper tract urothelial carcinomas: 2013 Update. Eur Urol. 63:1059–1071. 2013. View Article : Google Scholar : PubMed/NCBI | |
|
Edge SB and Compton CC: The American joint committee on cancer: The 7th edition of the AJCC cancer staging manual and the future of TNM. Ann Surg Oncol. 17:1471–1474. 2010. View Article : Google Scholar : PubMed/NCBI | |
|
Weng WH, Ahlén J, Aström K, Lui WO and Larsson C: Prognostic impact of immunohistochemical expression of ezrin in highly malignant soft tissue sarcomas. Clin Cancer Res. 11:6198–6204. 2005. View Article : Google Scholar : PubMed/NCBI | |
|
Gonzalez-Roibon ND, Chaux A, Al-Hussain T, Osunkoya AO, Bezerra SM, Hicks J, Epstein JI and Netto GJ: Dysregulation of mammalian target of rapamycin pathway in plasmacytoid variant of urothelial carcinoma of the urinary bladder. Hum Pathol. 44:612–622. 2013. View Article : Google Scholar : PubMed/NCBI | |
|
Rieken M, Shariat SF, Karam JA, Foerster B, Khani F, Gust K, Abufaraj M, Wood CG, Weizer AZ, Raman JD, et al: Frequency and prognostic value of PTEN loss in patients with upper tract urothelial carcinoma treated with radical nephroureterectomy. J Urol. 198:1269–1277. 2017. View Article : Google Scholar : PubMed/NCBI | |
|
Qian CN, Furge KA, Knol J, Huang D, Chen J, Dykema KJ, Kort EJ, Massie A, Khoo SK, Vanden Beldt K, et al: Activation of the PI3K/AKT pathway induces urothelial carcinoma of the renal pelvis: Identification in human tumors and confirmation in animal models. Cancer Res. 69:8256–8264. 2009. View Article : Google Scholar : PubMed/NCBI | |
|
Munari E, Fujita K, Faraj S, Chaux A, Gonzalez-Roibon N, Hicks J, Meeker A, Nonomura N and Netto GJ: Dysregulation of mammalian target of rapamycin pathway in upper tract urothelial carcinoma. Hum Pathol. 44:2668–2676. 2013. View Article : Google Scholar : PubMed/NCBI | |
|
Makboul R, Refaiy A, Abdelkawi IF, Hameed DA, Elderwy AA, Shalaby MM, Merseburger AS and Hussein MR: Alterations of mTOR and PTEN protein expression in schistosomal squamous cell carcinoma and urothelial carcinoma. Pathol Res Pract. 212:385–392. 2016. View Article : Google Scholar : PubMed/NCBI | |
|
Tsuruta H, Kishimoto H, Sasaki T, Horie Y, Natsui M, Shibata Y, Hamada K, Yajima N, Kawahara K, Sasaki M, et al: Hyperplasia and carcinomas in Pten-deficient mice and reduced PTEN protein in human bladder cancer patients. Cancer Res. 66:8389–8396. 2006. View Article : Google Scholar : PubMed/NCBI | |
|
Platt FM, Hurst CD, Taylor CF, Gregory WM, Harnden P and Knowles MA: Spectrum of phosphatidylinositol 3-kinase pathway gene alterations in bladder cancer. Clin Cancer Res. 15:6008–6017. 2009. View Article : Google Scholar : PubMed/NCBI | |
|
Koletsas N, Koletsa T, Choidas S, Anagnostopoulos K, Touloupidis S, Zaramboukas T, Raptou G, Papadopoulos N and Lambropoulou M: Immunohistochemical investigation of HER/AKT/mTOR pathway and cellular adhesion molecules in urothelial carcinomas. Patholog Res Int. 2017:67941502017.PubMed/NCBI | |
|
Brugge J, Hung MC and Mills GB: A new mutational AKTivation in the PI3K pathway. Cancer Cell. 12:104–107. 2007. View Article : Google Scholar : PubMed/NCBI | |
|
Franke TF, Kaplan DR, Cantley LC and Toker A: Direct regulation of the Akt proto-oncogene product by phosphatidylinositol-3,4-bisphosphate. Science. 275:665–668. 1997. View Article : Google Scholar : PubMed/NCBI | |
|
Kreisberg JI, Malik SN, Prihoda TJ, Bedolla RG, Troyer DA, Kreisberg S and Ghosh PM: Phosphorylation of Akt (Ser473) is an excellent predictor of poor clinical outcome in prostate cancer. Cancer Res. 64:5232–5236. 2004. View Article : Google Scholar : PubMed/NCBI | |
|
Vincent EE, Elder DJ, Thomas EC, Phillips L, Morgan C, Pawade J, Sohail M, May MT, Hetzel MR and Tavaré JM: Akt phosphorylation on Thr308 but not on Ser473 correlates with Akt protein kinase activity in human non-small cell lung cancer. Br J Cancer. 104:1755–1761. 2011. View Article : Google Scholar : PubMed/NCBI | |
|
Shin E, Choi CM, Kim HR, Jang SJ and Park YS: Immunohistochemical characterization of the mTOR pathway in stage-I non-small-cell lung carcinoma. Lung Cancer. 89:13–18. 2015. View Article : Google Scholar : PubMed/NCBI | |
|
Cha EK, Shariat SF, Kormaksson M, Novara G, Chromecki TF, Scherr DS, Lotan Y, Raman JD, Kassouf W, Zigeuner R, et al: Predicting clinical outcomes after radical nephroureterectomy for upper tract urothelial carcinoma. Eur Urol. 61:818–825. 2012. View Article : Google Scholar : PubMed/NCBI | |
|
Margulis V, Shariat SF, Matin SF, Kamat AM, Zigeuner R, Kikuchi E, Lotan Y, Weizer A, Raman JD and Wood CG; Upper Tract Urothelial Carcinoma CollaborationThe Upper Tract Urothelial Carcin, : Outcomes of radical nephroureterectomy: A series from the upper tract urothelial carcinoma collaboration. Cancer. 115:1224–1233. 2009. View Article : Google Scholar : PubMed/NCBI | |
|
Ehdaie B, Chromecki TF, Lee RK, Lotan Y, Margulis V, Karakiewicz PI, Novara G, Raman JD, Ng C, Lowrance WT, et al: Obesity adversely impacts disease specific outcomes in patients with upper tract urothelial carcinoma. J Urol. 186:66–72. 2011. View Article : Google Scholar : PubMed/NCBI | |
|
Izquierdo L, Truan D, Mengual L, Mallofré C and Alcaraz A: HER-2/AKT expression in upper urinary tract urothelial carcinoma: Prognostic implications. Anticancer Res. 30:2439–2445. 2010.PubMed/NCBI | |
|
Wheat JC, Weizer AZ, Wolf JS Jr, Lotan Y, Remzi M, Margulis V, Wood CG, Montorsi F, Roscigno M, Kikuchi E, et al: Concomitant carcinoma in situ is a feature of aggressive disease in patients with organ confined urothelial carcinoma following radical nephroureterectomy. Urol Oncol. 30:252–258. 2012. View Article : Google Scholar : PubMed/NCBI | |
|
Poon SL, Pang ST, McPherson JR, Yu W, Huang KK, Guan P, Weng WH, Siew EY, Liu Y, Heng HL, et al: Genome-wide mutational signatures of aristolochic acid and its application as a screening tool. Sci Transl Med. 5:197ra1012013. View Article : Google Scholar : PubMed/NCBI |
