Downregulation of CRTAC1 in Urothelial Carcinoma Promotes Tumor Aggressiveness and Confers Poor Prognosis

Background : Cartilage acidic protein 1 (CRTAC1) is a glycosylated calcium-binding extracellular matrix protein. The oncological functions of CRTAC1 in urothelial carcinoma (UC) of the urinary bladder (UB) and upper urinary tract (UT) have not yet been elucidated. Based on the published UBUC transcriptome data, we re-evaluated the differential expression profile of calcium ion binding–related genes (GO:0005509), and we found that CRTAC1 was the most significantly downregulated gene in UBUC progression. Therefore, we analyzed the prognostic value and biological significance of CRTAC1 expression in UC. Methods : We used immunohistochemistry to determine the CRTAC1 expression levels in 340 patients with UTUC and 295 patients with UBUC. The CRTAC1 expression was compared with the clinicopathological characteristics, and the prognostic impact of CRTAC1 on metastasis-free survival (MFS) and disease-specific survival (DSS) was evaluated. To study the biological functions of CRTAC1, the proliferation, migration, invasion, and tube formation abilities of UC-derived cells were evaluated. Results : A low CRTAC1 expression significantly correlated with high tumor stage, high histological grade, perineural invasion, vascular invasion, nodal metastasis, and high mitotic rate (all p < 0.01). Moreover, the CRTAC1 immunoexpression status was an independent prognostic factor for MFS and DSS in UBUC and UTUC patients (all p < 0.001) in the multivariate analysis. The exogenous expression of CRTAC1 suppressed the cell proliferation, invasion, and angiogenesis, and downregulated the matrix metallopeptidase 2 (MMP2) level in BFTC909 and T24 cells. Conclusions : CRTAC1 may participate in progression of UC and serve as a prognostic marker for metastasis. Low CRTAC1 expression was significantly associated with aggressive UC characteristics and worse clinical outcomes. The inclusion of CRTAC1 immunoexpression in the standard pathological variables may optimize the risk stratification of patients.


Introduction
Urothelial carcinomas (UCs) are the most common tumors of the urinary system diagnosed worldwide [1,2].They can be located in the urethra, urinary bladder, ureter, or renal pelvis.The majority of UC cases are urinary bladder UC (UBUC), while upper urinary tract UC (UTUC) accounts for only 5%-10% of UCs [3][4][5].The high histological and genetic heterogeneity of UC remains a clinical challenge [6,7].For UTUC, radical nephroureterectomy (RNU) is the standard treatment for high-risk diseases; kidneysparing surgery is recommended for patients with low risk UTUTC or serious renal insufficiency [5].For UBUC, most patients receive transurethral resection of the bladder tumor (TURBT) for non-muscle-invasive bladder cancer (NMIBC), followed by intravesical instillations [3].Radical cystectomy with perioperative chemotherapy is recommended for patients with muscle-invasive bladder cancer (MIBC) or high risk NMIBC [3,4].Advances in therapeu-tic modalities, surgical techniques, and health care systems have improved disease management strategies.However, the overall prognosis remains unsatisfactory [2][3][4][5].Therefore, understanding the mechanisms underlying UC progression is critical for improving patient stratification and disease management.
Calcium (Ca 2+ ) is a secondary messenger that regulates several diverse biological and pathological processes in cells [8].Dysregulation of calcium signaling is associated with tumorigenesis and cancer progression [9].Some cellular functions are mediated by calcium-binding proteins [10].Several calcium-binding proteins are known to play essential roles for urinary calcium oxalate stone formation processes [11].However, the roles of calcium-binding proteins in the development of UC have not been investigated much.To better understand the significances of calciumbinding proteins in UC tumorigenesis, we used a public UC transcriptomic dataset (GSE32894) to investigate the differentially expressed genes (DEGs) related to the molecular function of calcium ion binding (GO:0005509).CR-TAC1 (cartilage acidic protein 1) was the most downregulated gene associated with UC progression.
CRTAC1, which is a glycosylated extracellular matrix (ECM) protein, is found in human articular cartilage [12].Arg-Gly-Asp (RGD) integrin-binding motifs and Phe-Gly (FG) with Gly-Ala-Pro (GAP) (FG-GAP) motifs play significant roles in cell-matrix and cell-cell interactions [11].In human dermal fibroblasts, CRTAC1 is involved in ECM development; organization, remodeling, and degradation of collagen; wound healing; and cellular regeneration, migration, and proliferation [13,14].In human lens epithelial cells, upregulation of CRTAC1 promotes ultraviolet B-induced pyroptosis and cataract formation via reactive oxygen species signaling [15,16].In osteoarthritis patients, CRTAC1 is an important regulator and its expression is induced by upregulation of IL-1β and TNF-α, resulting in the promotion of catabolism and inhibition of the anabolic activities of chondrocytes [17].In bladder cancer, Yang et al. [18] demonstrated that CRTAC1 inhibited cell proliferation, migration, and invasion by targeting Yin Yang 1 to inactivate the TGF-β pathway.He et al. [19] found TFAP2A promoted TPRG1-AS1 transcription to reduce CRTAC1 expression, thereby accelerating bladder cancer cells glycolysis and angiogenesis.Using public bladder cancer datasets, Wang et al. [20] constructed a prognostic model based on the cuproptosis subtype-related prognostic differentially expressed genes, which included eight gene predictors (PDGFRB, COMP, GREM1, FRRS1, SDHD, RARRES2, CRTAC1, and HMGCS2).These studies only evaluated the function of CRTAC1 in UBUC, the possible role of CRTAC1 in UTUC has not yet been elucidated.Therefore, we assessed the prognostic values of CRTACI in UTUC and UBUC, and we investigated the functions and mechanisms of CRTACI in UC progression.

Data Mining
We performed transcriptomic profiling of a Gene Expression Omnibus (GEO) dataset (GSE32894) composing of 308 UBUC patients [21].All probes without filtering or preselection were analyzed, and the raw data were imported into the Nexus Expression 3.0 (BioDiscovery, El Segundo, CA, USA) to compute gene abundances, as described in our previous study [22].A comparative analysis (noninvasive vs. invasive UC) was performed to examine the DEGs related to calcium ion binding (GO:0005509).Those with a significant log 2 -transformed expression fold change <-0.15 (p < 0.01) were selected for further analysis.

Study Population
A total of 340 UTUC and 295 UBUC patients who underwent curative surgery between 1998 and 2004 were enrolled in the study, and all specimens were procured from our biobank after obtaining informed consent.The patients' clinical demographic characteristics, pathological features, and survival outcomes were retrospectively reviewed from their medical charts.None of the patients had undergone preoperative radiotherapy or chemotherapy.Postoperative platinum -based adjuvant chemotherapy was administered to patients with nodal involvement or pT3-pT4 diseases.The pathological grade and tumor stage were determined based on the WHO classification criteria and the 7th edition of the american joint committee on cancer (AJCC) staging system, respectively.The Institutional Review Board of Chi Mei Medical Center approved this study (10501005).

Immunohistochemical Assessments
Paraffin-embedded tissue blocks were sliced (4 µm) and placed on silane-coated slides, as previously described [22].Deparaffinization, rehydration, and antigen retrieval were performed according to the standard procedures.The endogenous peroxidase activity was blocked using 3% H 2 O 2 (ab64218, Abcam, Cambridge, England).The slides were then incubated with primary antibodies against CR-TAC1 (1:100, ab254691, Abcam, Cambridge, England), matrix metallopeptidase 2 (MMP2) (1:100, ab86607, Abcam), or CD31 (1:100, ab28364, Abcam) for one hour.The target proteins were detected using a ChemMate™ EnVi-sion™ Kit (Dako, Carpinteria, CA, USA).Positive CR-TAC1 expression was characterized by membranous and/or cytoplasmic staining in UC cells.Sections processed without the primary anti-CRTAC1 antibody were used as negative controls.Two independent pathologists estimated the cancer cell distribution and the intensity of the immunohistochemical (IHC) staining to generate an H-score using the formula ΣPi(i + 1), where Pi is the percentage of stained cancer cells (0%-100%) and i is the intensity of the stained cancer cells (0-3+).The immunoreactivity of CRTAC1 was described as low or high levels of expression according to the median H-score,.As previously described, we iden-tified the CD31-labeled vessels and used ImageJ software to calculate the tumoral microvessel density (MVD) [23].

Cell Culture
Five human UC-derived cell lines, namely TCCSUP (American Type Tissue Culture Collection [ATCC], VA), J82 (ATCC, VA), T24 (ATCC, VA), BFTC905 (Food Industry Research and Development Institute [FIRDI], Taiwan), and BFTC909 (FIRDI, Taiwan), were screened for CRTAC1 expression.A non-tumoral uroepithelial cell line, SV-HUC-1 (ATCC, VA), was used as a control.BFTC909 and T24 cells, which exhibit relatively low levels of endogenous CRTAC1, were selected for this study.These cells were cultured as previously described [24].All cells were incubated at 37 °C in a humidified incubator containing 5% CO 2 .The cell lines were regularly tested for mycoplasma and were authenticated by short tandem repeat genotyping.

Exogenous CRTAC1 Overexpression in T24 and BFTC909 Cells
The Phoenix-Amphotropic (AMPHO) cell line (ATCC, VA) was used to produce lentiviral particles containing CRTAC1.Briefly, the expression plasmids for CRTAC1 and the control vector (pLenti-GIII-CMV) were purchased from Applied Biological Materials, Inc (Richmond, BC, Canada).Following a series of transfections, as described previously [24], T24 and BFTC909 were incubated with culture medium containing viral supernatant and 10 µg/mL polybrene for 24 hours.Afterward, medium containing viral solution was replaced with fresh medium.UC cell lines overexpressing CRTAC1 were obtained as stable clones after puromycin selection (2 µg/mL).

Real-Time Quantitative RT-PCR
Total RNA was extracted from the UC cells using a Quick-RNA™ Miniprep Kit (Zymo Research, Beijing, China), and was reverse-transcribed using a Maxima First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA).The cDNA was mixed with the corresponding TaqMan assay probes (CRTAC1, Hs00907892_m1; MMP2, Hs01548727_m1; MMP9, Hs00957562_m1; POLR2A, Hs01108291_m1; Applied Biosystems) using TaqMan™ Fast Advanced Master Mix (Thermo Scientific).We then performed quantitative RT-PCR for the mRNA level by the 2 −∆∆CT method using a StepOnePlus™ System (Applied Biosystems, Waltham, MA, USA).The relative expression levels of the target mRNAs were normalized to those of POLR2A RNA.

Western Blot Assays
We used PRO-PREP™ Protein Extraction Solution (iNtRON Biotechnology, Seongnam-Si, Gyeonggi-do, Republic of Korea) to extract total cellular proteins.The protein concentration was determined by a BCA assay kit (Thermo Fisher Scientific).Thirty micrograms of protein were loaded on an mPAGE Bis-Tris Precast Gel (Merck Millipore, Burlington, MA, USA) and transferred onto an Immobilon®-P PVDF membrane (Merck Millipore).The PVDF membranes were blocked with 5% skim milk (Millipore Sigma, Hong Kong, China) incubated overnight at 4 °C with the following primary antibodies: anti-CRTAC1 (ab254691, Abcam), anti-MMP2 (ab86607, Abcam), or anti-GAPDH (ab181602, Abcam).GAPDH served as an internal control.After washing, the membrane was incubated with a diluted secondary antibody (horseradish peroxidase [HRP] donkey anti-rabbit immunoglobulin G, Bi-oLegend) for one hour.CRTAC1, MMP2, and GAPDH were visualized by enhanced chemiluminescence (Thermo Scientific).

Cell Proliferation Assay
Cell proliferation was evaluated using a Cell Proliferation Assay Kit (Fluorometric; BioVision, Hong Kong, China).Briefly, 1000 cells were pleated in 96-well microplates and incubated for 24, 48, and 72 h at 37 °C.Subsequently, 25 µL of the reaction mixture, including 1× nuclear dye/cell lysis buffer solution and 1× nuclear dye, was added to each well.After 15 min of incubation, the fluorescence intensity was determined using a standard microplate reader (excitation/emission = 480/538 nm).The assays were repeated at least three times.

Migration and Invasion Assays
The Boyden chamber technique (Transwell® analysis, Thermo Scientific, Hong Kong, China) was used to determine the cell migration and invasion abilities [25].Cell migration and invasion assays were performed using Falcon HTS FluoroBlok 24-well inserts (BD Biosciences, Franklin Lakes, NJ, USA) and a QCM™ Collagen Cell Invasion Assay (Millipore), respectively.Each insert was rehydrated with serum-free medium and 1 × 10 6 UC cells suspended in serum-free medium were plated in the upper chamber and incubated with medium containing 10% FBS in the lower chamber.After 12-24 h of incubation, migrating and invading cells passing through the inserts were detached, stained with the provided dye and transferred to 96-well plates for colorimetric analysis at 560 nm.The assays were repeated at least three times.

In Vitro Tube Formation Assay
Human umbilical vein endothelial cells (HUVECs) were used to investigate the effects of CRTAC1 on UCinduced angiogenesis.The Matrigel® Matrix (Corning, Tewksbury, MA, USA) was used to precoat each inner well of the µ-Slide Angiogenesis (Ibidi, Gräfelfing, Germany) for 30 min at 37 °C.Then, 50 µL of cell suspension containing 7 × 10 3 HUVECs in 25 µL conditioned medium and 25 µL endothelial cell medium with 2% FBS was seeded on top of the Matrigel®.After incubation for 5 h at 37 °C, the capillary-like tube structures were evaluated and counted under a phase contrast microscope.The assays were repeated at least three times.

Luciferase Reporter Assays
The vector pcDNA3-CRTAC1 was constructed as previously described [26].MMP2 and MMP9 promoter fragments were cloned by PCR amplification and inserted into a luciferase reporter gene plasmid vector (Promega, Fitchburg, WI, USA).The MMP2 and MMP9 promoter reporter constructs and pcDNA3-CRTAC1 construct or their matching empty constructs were co-transfected in cells using PolyJet TM transfection reagent (SignaGen Laboratories, Shandong, China) for 48 h.We then used a Dual-Glo® Luciferase Reporter Assay System (Promega) to measure the luciferase activity following the manufacturer's protocol.

Statistical Analyses
Associations between CRTAC1 expression and different variables were evaluated using the chi-square test.For survival analyses, the disease-specific survival (DSS) and metastasis-free survival (MFS) were plotted using Kaplan-Meier curves and estimated using the log-rank test at the univariate level.The independent prognostic factors were estimated using a multivariate Cox proportional hazards model.All cellular functional studies were done with three replicates.Student's t-test was used to analyze differences in cell proliferation, migration, invasion, HUVEC tube formation, and luciferase activity.Statistical analyses were performed using SPSS 19 software (SPSS Inc., Chicago, IL, USA).In all analyses, a p-value of <0.05 was considered to reflect statistical significance.

CRTAC1 is the Most Significantly Downregulated Gene Related to the Calcium Ion Binding in UBUC Invasiveness
We identified 34 probes covering 22 transcripts associated with calcium ion binding (GO:0005509) in UBUC invasion using a published transcriptome dataset (GSE32894).These genes were significantly downregulated at the high tumor stage (Fig. 1 and Table 1).CRTAC1 was selected for further evaluation because it was the most downregulated gene.Using the Gene Expression Profiling Interactive Analysis database, CRTAC1 was found to be significantly decreased in UBUC (n = 404) compared to in adjacent normal tissues (n = 19) (p < 0.001).Intriguingly, violin plots showed that CRTAC1 mRNA levels significantly decreased as the UBUC stage increased (from stage II to IV).Moreover, UBUC patients with high CRTAC1 mRNA expression had better overall survival than those with low CRTAC1 mRNA expression (p = 0.0007) (Supplementary Fig. 1).These results prompted us to assess the prognostic value and clinical relevance of CRTAC1 in a large cohort of UC patients.

Prognostic Significance of CRTAC1 Expression
Within a median follow-up of 31.7 months, 70 UTUC and 76 UBUC patients developed tumor metastasis, while 61 UTUC and 52 UBUC patients died of UC.

CRTAC1 Inhibition of UC Cell Proliferation, Invasion, and Angiogenesis
To understand the biological function of CRTAC1, endogenous CRTAC1 expression in UC cell lines was determined.Compared to normal urothelial primary cells (SV-HUC-1), all five UC-derived cell lines had lower CR-TAC1 mRNA and protein expression (Fig. 4A).Of these, BFTC909 and T24 cells exhibited the lowest levels of CR-TAC1 expression; therefore, CRTAC1 overexpression was induced in these two cell lines (Fig. 4B).The overexpression of CRTAC1 in BFTC909 and T24 cells significantly attenuated cell proliferation (Fig. 4C).Matrigel® invasion assays indicated that CRTAC1 overexpression also significantly decreased the number of invading tumor cells, thus indicating its ability to inhibit metastasis (Fig. 4D).More- over, conditioned medium from CRTAC1-overexpressing BFTC909 and T24 cells markedly inhibited HUVEC tube formation compared to that in the mock group (Fig. 5A).In addition to in vitro studies, we studied the association between CRTAC1 and MVD in our UC specimens.Notably, high CRTAC1 expression was significantly correlated with less CD31-labeled MVD in UTUC and UBUC.(Fig. 5B) To identify the potential cellular pathways that are involved in the regulation of UC invasiveness by CRTAC1, MMP2 was selected for further studies.Initially, IHC staining showed that high CRTAC1 expression negatively correlated with low MMP2 expression in UTUC and UBUC (Table 2; Fig. 2).qRT-PCR and immunoblotting showed that exogenous CRTAC1 expression markedly suppressed MMP2 mRNA and protein expression in BFTC909 and T24 cells (Fig. 6A).Finally, analysis of luciferase activity driven by the MMP2 promoter showed that MMP2 transactivation was negatively associated with CRTAC1 expression in UC cells.These results confirm the role of MMP2 in CRTAC1driven UC aggressiveness (Fig. 6B).

Discussion
UCs, including UTUC and UBUC, have genetic and clinical heterogeneity [6,7].Despite the advances in treatment modalities and surgical techniques, patient survival rates remain poor.Therefore, the incorporation of genetic information may optimize the risk stratification of patients and disease management.Through transcriptomic profiling, we discovered that CRTAC1 was the most downregulated calcium-ion-binding gene in UC.In the The Cancer Genome Atlas (TCGA) bladder cancer database, CR-TAC1 mRNA abundance in cancer tissues was lower than that in adjacent normal tissues.Its expression was notably decreased in patients with high stage cancer.These observations suggest that CRTAC1 acts as a tumor suppressor during UC progression.Accordingly, the clinical relevance of CRTAC1 was evaluated in our well-characterized UC cohorts.CRTAC1 expression was an independent prognostic factor for MFS and DSS, after adjusting for important pathological parameters.Patients with high CRTAC1 expression had significantly better clinical prognosis.Our study was the first to report an association between CR-TAC1 expression and metastasis and survival in UBUC and UTUC.
CRTAC1, located on chromosome 10q24.2,encodes a glycosylated calcium-binding ECM protein called cartilage acidic protein 1 [12].However, the functions of CR-TAC1 reamin poorly understood.It contains a calciumbinding epidermal growth factor domain and an integrin alpha chain-like domain that interacts with ECM proteins and mediates cell-cell and cell-matrix interactions [12,27,28].This protein was originally discovered during the chondrocyte development, and its concentration was relatively high in patients with osteoarthritis [11,16].CRTAC1 also promotes apoptosis and pyroptosis in human lens epithelial cells resulting in cataract formation [15,29].It regulates energy metabolism and promotes proliferation and migration in primary human dermal fibroblasts [14].However, the role of CRTAC1 in UC tumorigenesis and progression remains unclear.Accordingly, its prognostic significance in large UBUC and UTUC cohorts was evaluated.
In UBUC, most patients with NMIBC underwent TURBT, intravesical instillations, and cystoscopic assessments after the survey [3].Progression to high-grade or detrusor muscle invasive tumors is a critical issue in NMIBC management.In this study, low CRTAC1 immunoexpression correlated with high tumor grade and stage in patients with UBUC, suggesting that CRTAC1 is a potential marker of UC invasiveness.Early radical cystectomy may be advantageous for patients with NMIBC with low CR-TAC1 expression.Multimodal bladder preservation treatment has been suggested for highly selected patients with MIBC [4,30].Patients with low CRTAC1 expression in MIBC, who have a high risk of distant organ and nodal metastases, may require radical surgery.The inclusion of CRTAC1 expression in pathological parameters may help physicians select suitable candidates for bladder-preserving treatments.
UTUC is a rare genitourinary disease, accounting for 5%-10% of new UC cases.At the time of diagnosis, 60% of UTUC cases are considered invasive compared to 25%  of UBUC cases [2,5].Therefore, current guidelines recommend RNU as the standard treatment for patients with high-grade UTUC [5].However, the benefits of lymph node dissection and its optimal extent have not yet been determined in non-metastatic UTUC.In this study, low CRTAC1 expression significantly correlated with perineural invasion, vascular invasion, and nodal metastasis, resulting in poor clinical outcomes.If RNU with lymph node dissection is recommended for patients with low CRTAC1 expression, those with UTUC may be suitable candidates for this treatment.These patients are also good candidates for adjuvant chemotherapy because of the significantly high probability of subsequent metastasis.Owing to the old age, renal insufficiency, and medical comorbidities of UTUC patients, kidney-sparing management is recommended for patients with low-grade or low-stage disease [14,31].However, accurate preoperative tumor staging is challenging.Ureteroscopic biopsy makes it difficult to obtain adequate tissue to assess invasion depth.Based on our results, aggressive features can be determined by assessing the CRTAC1 immunoexpression status in biopsy specimens.Additional information can also help achieve optimal decision making.
Letsiou et al. [13] elucidated the functions of CRTAC1 in tissue biology by performing high-throughput RNA sequencing transcriptome analysis.These results demonstrated that CRATC1 regulates ECM organization in the complement cascade.The molecular mechanisms of CR-TAC1 bioactivity in the wound healing process have been investigated in primary human dermal fibroblasts [14].Gene expression analysis revealed that the CRTAC1 protein was associated with cell proliferation (downregulated CXCL12 and upregulated NOS2), cell migration (upregulated AQP3 and downregulated TNC), and extracellular matrix regeneration and remodeling (upregulated FMOD, upregulated TIMP1, downregulated FN1, and downregulated COL3A1).Similar altered genes have been found in a zebrafish skin damage repair model [32].However, the biology of CRTAC1 in cancer remains unclear.In lung cancer, CRTAC1 expression in cancer tissues was lower than in normal tissues.Yu et al. [33] developed a 5-gene (KRT6A, MELTF, IRX5, MS4A1 and CRTAC1) signature prognostic stratification system to predict the overall survival of patients with lung cancer.In gastric cancer, low expression of CRTAC1 was strongly associated with a poor prognosis.Shen et al. [34] constructed a 8-gene (KCNJ2, GATA5, CLDN1, SERPINE1, FCER2, PMEPA1, TMEM37 and CRTAC1) survival prognosis model.They found highrisk group is more likely to escape immunity and less sensi-tive to immunotherapy and chemotherapy [34].In bladder cancer, Yang et al. [18] found that CRTAC1 overexpression inhibited cell proliferation, viability, migration, invasion and epithelial-mesenchymal transition process by downregulating Yin Yang 1 to inactivate the TGF-β pathway.In the present study, we assessed the biological functions of CRTAC1 in UC-derived cell lines.Similar to human tissues, CRTAC1 expression in UC cells was lower than that in non-tumoral urothelium.Cell proliferation, Transwell® assays, HUVEC tube formation, and immunoblotting assays showed that CRTAC1 overexpression inhibited cell proliferation, invasion, angiogenesis, and MMP2 expression.These findings are in accordance with our observation that high CRTAC1 expression was associated with low tumor stage/grade, low MVD, low incidence of UC metastasis, and better survival in our clinical cohort.

Conclusions
CRTAC1 expression decreases during the transition from normal urothelium to superficial and invasive UC, indicating its potential role in carcinogenesis and invasiveness.In addition, the exogenous overexpression of CR-TAC1 attenuated UC-derived cell line proliferation, invasion, and angiogenesis.Therefore, CRTAC1 is a promising therapeutic target for UC.The present study also demonstrated the independent prognostic importance of CRTAC1 in survival and metastasis risk in patients with UBUC and UTUC.Therefore, close surveillance and aggressive treatments are crucial for patients with UC and low CRTAC1 levels.The addition of CRTAC1 immunostaining to routine histopathological examinations can help clinicians identify high-risk patients and facilitate individualized therapy.

Fig. 1 .
Fig. 1.Data mining.Expression profiles of genes associated with the calcium ion binding (GO:0005509) extracted from a published transcriptome of UC (GSE32894) in the Gene Expression Omnibus database.CRTAC1 was the most downregulated gene associated with UC progression.UC, urothelial carcinoma.

Fig. 3 .
Fig. 3. Kaplan-Meier survival curves.Low CRTAC1 expression is associated with a significant prognostic impact on diseasespecific survival and metastasis-free survival of patients with UTUC (A and B, respectively) and UBUC (C and D, respectively).UTUC, upper urinary tract urothelial carcinoma; UBUC, urinary bladder urothelial carcinoma.

Fig. 4 .
Fig. 4. CRTAC1 expression inhibits growth and invasion of UC cells in vitro.(A) Compared to SV-HUC-1 cells, endogenous CRTAC1 mRNA and protein expression is lower in T24 and BFTC909 cell lines.(B) CRTAC1 overexpression was induced in these two cell lines.Compared with lentiviral infection with the mock sequence, lentiviral infection with CRTAC1 significantly increased the mRNA and protein levels of CRTAC1 in BFTC909 and T24 cells.(C) The overexpression of CRTAC1 in BFTC909 and T24 cells significantly attenuates the cellular proliferation.(D) Using Transwell® migration and invasion assays, cell invasion is significantly reduced in CRTAC1-transfected T24 and BFTC909 cell lines, compared to that in the corresponding empty controls.(*, p < 0.05).

Fig. 5 .
Fig. 5. CRTAC1 expression inhibits angiogenesis of UC. (A) Tube formation is markedly decreased when the HUVECs are incubated with conditioned medium from the CRTAC1-overexpressing T24 and BFTC909 cells than that from the mock groups.(B) High CRTAC1 expression is significantly correlated with less CD31-labeled microvascular density in UTUC and UBUC.(*, p < 0.05).

Fig. 6 .
Fig. 6.CRTAC1 inhibits UC invasion by transcriptional repression of MMP2.(A) Exogenous CRTAC1 expression significantly downregulated the MMP2 mRNA and protein levels in BFTC909 and T24 cells, using qRT-PCR and western blotting.(B) The luciferase activity of MMP2 promoter construct was significantly lower in the T24 and BFTC909 cells transfected with the CRTAC1-expressing vector than that in the empty controls.(*, p < 0.05).

Table 3 . Univariate log-rank and multivariate analyses for disease-specific and metastasis-free survivals in upper urinary tract urothelial carcinoma.
* Statistically significant.HR, hazard ratio; CI, confidence interval.