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Enhanced expression of caveolin-1 possesses diagnostic and prognostic value and promotes cell migration, invasion and sunitinib resistance in the clear cell renal cell carcinoma
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Enhanced expression of caveolin-1 possesses diagnostic and prognostic value and promotes cell migration, invasion and sunitinib resistance in the clear cell renal cell carcinoma HaiLong Ruana,1, Xiang Lia,1, HongMei Yangb, ZhengShuai Songa, JunWei Tonga, Qi Caoa, KeShan Wanga, Wen Xiaoa, HaiBin Xiaoa, XuanYu Chena,c,d, GuangHua Xua, Lin Baoa, ZhiYong Xionga, ChangFei Yuana, Lei Liua, Yan Qub, WenJun Hub, YaoYing Gaob, ZeYuan Rub, ⁎ Ke Chena, XiaoPing Zhanga, a Department of Urology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1277 Jiefang Avenue, Wuhan 430022, Hubei Province, China b Department of Pathogenic Biology, School of Basic Medicine, Huazhong University of Science and Technology, 13 Hangkong Road, Wuhan 430030, Hubei Province, China c Department of Urology, Zhejiang Provincial People's Hospital, Hangzhou 310014, China d Department of Neuroscience and Regenerative Medicine, Medical College of Georgia, Augusta University, Augusta, GA 30912, USA
A R T I C L E I N F O
A BS T RAC T
Keywords: CAV1 Sunitinib-resistance Invasion Prognosis Diagnosis ccRCC
Caveolin-1 (CAV1) has been identified to be up-regulated in many cancers, including clear cell renal cell carcinoma (ccRCC). However, its potential function is still unclear in ccRCC. In this study, we demonstrated that CAV1 was frequently overexpressed in renal cell carcinoma tissues and cells, and was significantly associated with various clinicopathological parameters. In addition, high CAV1 expression was associated with poor disease-free survival (DFS) rate and could serve as a useful diagnostic indicator in ccRCC patients with different clinicopathological stages. Functional experiments demonstrated that CAV1 knockdown inhibited cell migration and invasion, whereas overexpression of CAV1 promoted cell migration and invasion in ccRCC. Moreover, CAV1 expression was up-regulated in sunitinib-resistant renal cancer cell lines, and its overexpression promoted sunitinib resistance. In general, our results confirm that CAV1 plays an important role in the metastasis of kidney cancer and induces sunitinib resistance, so CAV1 function suppression may become a promising clinical treatment strategy during renal cell carcinoma metastasis and sunitinib resistance.
1. Introduction Renal cell carcinoma (RCC) is the most common malignant neoplasm in the kidney, which accounts for approximately 80–90% renal malignancies and about 2–3% adult malignancies. It is predicted that approximately 63,990 new patients of renal cancer and 14,400 deaths from renal cancer will emerge in the U.S in 2017 [1]. RCC comprises a variety of different pathologic subtypes. Clear cell RCC (ccRCC) is the most common subtype that is generally accompanied by high rate of metastasis and mortality, and nonsensitive to conventional chemotherapy and radiotherapy. Tumor metastasis is the leading cause of death in cancer-bearing patients [2]. Prompt surgical treatment for localized RCC patients can achieve a good prognosis and survival rate. Disappointingly, approximately 30% of patients with RCC have pro⁎
1
gressed into local invasion or distant metastasis when clinically diagnosed, leading to poor prognosis. In recent years, receptor tyrosine kinase inhibitors (RTKI), including sunitinib, have become first-line therapeutic agents for metastatic and advanced RCC. However, due to intrinsic or acquired resistance to RTKI and high side effects of drugs, most RCC patients are restricted to use these drugs. Therefore, it is urgently needed to explore the metastatic and RTKI-resistant mechanisms in RCC and develop new therapeutic drugs. Caveolins are structurally and functionally evolutionarily conserved and represent the major components of caveolae, which are specialized lipid raft microdomains of the cytoplasmic membrane involved in intracellular signal transduction pathways that regulate cell growth, adhesion and survival [3]. Caveolin-1 (CAV1), belonging to the caveolin supergene family, is originally identified as a 24 kDa protein that form
Corresponding author. E-mail address: [email protected] (X. Zhang). These authors contribute equally to this study.
http://dx.doi.org/10.1016/j.yexcr.2017.07.004 Received 11 June 2017; Received in revised form 29 June 2017; Accepted 1 July 2017 0014-4827/ © 2017 Published by Elsevier Inc.
Please cite this article as: Ruan, H., Experimental Cell Research (2017), http://dx.doi.org/10.1016/j.yexcr.2017.07.004
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Fig. 1. The level of CAV1 is upregulated and correlated with various clinicopathological parameters in ccRCC tissues. (A-F) The mRNA level of CAV1 in ccRCC was downloaded from the TCGA_KIRC dataset containing 72 para-cancer tissues and 533 ccRCC tissues. The mRNA levels of CAV1 were compared in different clinicopathological parameters: (A) cancer versus para-cancer, (B) gender, (C) T stage, (D) TNM stage, (E) M stage, (F) G stage.
oncogenic and drug-resistant gene in RCC tumor microenvironment and provided a firm theoretical principle for consideration of CAV1 combining with receptor tyrosine kinase inhibitors as a new therapy for metastatic and advanced RCC.
caveolae at the plasma membrane. Dysregulation of CAV1 expression is often associated with tumorigenesis, progression, prognosis, metastasis, multidrug- resistance [4–8]. CAV1 was upregulated in prostate cancer [7], bladder cancer [9], pancreas cancer [10]. On the other hand, CAV1 was downregulated in colon cancer [11], breast cancer [12]. Thus, the biological role of CAV1 in tumor cells is quite complicated depending on the origin and the microenvironment of the tumor. However, few studies have been carried out to investigate correlation between CAV1 expression and RTKI-resistance in renal cell carcinoma and the biological behavior of CAV1 in renal cancer cells. In our research, we for the first time demonstrated that CAV1 is significantly upregulated in sunitinib-resistance RCC cells and promotes sunitinib resistance in RCC cells. Moreover, we identified CAV1 was overexpressed in RCC cells and tissues, and promoted cell migration and invasion. These data indicated that CAV1 might be an
2. Materials and methods 2.1. Renal cancer tissue samples We collected 60 pairs of ccRCC and adjacent normal renal tissues from nephrectomies operated at the Wuhan Union Hospital between 2015 and 2016. Resected tissues were divided into two halves: one half was promptly frozen in liquid nitrogen for subsequent western blot experiments; the other half was fixed with formalin and paraffinembedded for matched immunohistochemistry assays. Before surgery, 2
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all patients did not receive any chemotherapy or radiotherapy. All surgical patients had signed an informed consent form. The study and experimental procedures were approved by the Human Research Ethics Committee of Huazhong University of Science and Technology.
Table 1 Correlation between CAV1 mRNA expression and clinicopathological parameters of ccRCC patients. CAV1 mRNA expression Parameter
Age (years) Gender T stage N stage M stage G stage TNM stage
< = 60 > 60 Male Female T1 + T2 T3 + T4 N0 + NX N1 M0 + MX M1 G1 + G2 + GX G3 + G4 I + II III + IV
Number
Low (n = 263)
High (n = 263)
261 265 341 185 337 189 510 16 448 78 247 279 319 207
128 135 147 116 179 84 257 6 238 25 136 127 174 89
133 130 194 69 158 105 253 10 210 53 111 152 145 118
P value
2.2. Cell culture and drug intervention The human renal cancer cell lines 786-O, ACHN, A498, Caki-1, OSRC-2 and renal tubular epithelial cell line HK-2 were purchased from The American Type Culture Collection (ATCC, USA). Cells were maintained in DMEM high glucose medium (Google Biotechnology Co., Ltd, Wuhan, China) containing 10% FBS (Lilac Garden Technology Co., Ltd, Wuhan, China) and 1% penicillin-streptomycin at 37 ℃ in a 5% CO2 incubator. For drug intervention, sunitinib (Pfizer) was dissolved in DMSO at a concentration of 15 mg/ml and then added to the culture medium of renal cancer cells at the indicated concentrations.
0.663 0.000 0.056 0.310 0.001 0.029
2.3. Establishment of sunitinib-resistant cell lines 0.010
To establish sunitinib-resistant renal carcinoma cell lines, 786-O and ACHN were established by continuous low-dose stimulation in
Fig. 2. High level of CAV1 mRNA predicts poor disease-free survival rate in ccRCC patients. (A) The ccRCC patients from TCGA-KIRC database were divided into low CAV1 expression group and high CAV1 expression group according to the median expression value of CAV1 mRNA level. The correlation between CAV1 expression and disease-free survival time of total ccRCC patients was analyzed by Kaplan-Meier. (B-I) Disease-free survival analysis towards the expression of CAV1 mRNA was performed in subgroups of ccRCC patients: (B) T1 + T2 stage, (C) T3 + T4 stage, (D) N0 stage, (E) M0 stage, (F) G3 + G4 stage, (G) Male, (H) Age ≤ 60 years, (I) Age > 60 years.
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Fig. 3. High CAV1 expression serves as a diagnostic indicator in ccRCC patients. (A) ROC curve showed that CAV1 could effectively distinguish ccRCC from para-cancer tissues. The AUC was 0.9515 (p < 0.0001). (B-F) ROC curve analysis towards the expression of CAV1 mRNA in subgroups of ccRCC patients against TNM stage (B), T stage (C), M stage (D), G stage (E) and DFS status (F).
2.5. Immunohistochemistry assay
conjunction with intermittent high-dose pulses of sunitinib as described and characterized [13]. The IC50 values of the sunitinibresistant cell lines are more than twice that of the parental cell lines.
Briefly, ccRCC tissues and corresponding para-cancer tissues were formalin-fixed, dehydrated, and paraffin-embedded. The tissue sections were incubated with rabbit CAV1 polyclonal antibody (1:100, Santa Cruz, CA, USA) overnight at 4 ℃. The next day, tissue sections were washed three times with PBS and then incubated with horseradish peroxidase conjugated secondary antibody.
2.4. Transient transfection assay The si-RNA oligonucleotide sequences targeting CAV1 (si-CAV1) and si-RNA Negative Control (si-NC) were synthesized by Gene-Pharma (ShangHai, China). BLAST alignment of all si-RNA sequences was performed to ensure that they had no off-target effect. The si-CAV1 and si-NC with a final concentration of 50 nM were transfected with lipofectamine 2000 (Invitrogen, USA) according to the manufacturer's protocols. The CAV1 plasmid (CAV1) and Negative Control vector (Vector) were purchased from Vigene Biosciences (Shan Dong, China). A total 2 μg of CAV1 or Vector plasmid were transfected with lipofectamine 2000 according to the manufacturer's recommendations. Cells were collected for subsequent assays at 48 h after transfection. The si-CAV1 sequences were shown as following: #1: 5′-CGACGTGGTCAAGATTGACTT-3′; #2: 5′GCTTCCTGATTGAGATTCAGT-3′;
2.6. Immunofluorescence The appropriate numbers of cells were planted on glass coverslips, washed three times with PBS, fixed in 4% paraformaldehyde for 10 min. Cells were blocked for 1 h with 3% BSA and then were incubated with CAV1 primary antibodies overnight at 4 ℃. The next day, these cells were incubated for 1 h with CY3-conjugated secondary antibodies, and then stained with DAPI. Finally, these cells were photographed under fluorescence microscopy.
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Fig. 4. CAV1 expression is elevated in our ccRCC cells and tissues. (A-B) Western blot analysis of CAV1 expression in renal cancer cell lines and ccRCC tissues. (C) Immunohistochemistry (IHC) analysis of CAV1 expression in ccRCC tissues and paracancer tissues. Representative images were shown.
2.7. Wound healing assays and transwell invasion assays For wound healing assays, cells transfected with si-CAV1 and si-NC were seeded in 6-wells plate. After reaching 80–90% confluent, the cells were scratched with 10-μl pipette tips, washed, and then photographed at 0 h and 24 h. In vitro cell invasion assays were carried out using 24-well transwell chambers with 8.0 µm pore size (Corning, NY, USA). For the invasion assay, 2 × 104 (786-O, ACHN) cells transfected with si-CAV1 and si-NC were added to the upper chamber of the transwell chamber precoated with Matrigel (BD Biosciences, NJ, USA). The medium containing 10% FBS was added to the lower chamber. After 24 h incubation, invaded cells were fixed with methanol and then stained with 0.05% crystal violet. The invaded cells were counted in 5 random fields.
Fig. 5. The sunitinib sensitivity curve of sunitinib-resistant and parental cells. (A) The sunitinib sensitivity curve of 786-O and 786O-R. (B) The sunitinib sensitivity curve of ACHN and ACHN-R. (C) IC50 value of sunitinib-resistant and parental cells. The error bars represent mean ± SD of three independent experiments (**, P < 0.01, *, P < 0.05, compared with the corresponding parental cells).
GAPDH Forward: 5′-GGTGAAGGTCGGAGTCAACGG-3′; GAPDH Reverse: 5′-GAGGTCAATGAAGGGGTCATTG-3′.
2.8. Quantitative real-time PCR assays (qRT-PCR)
2.9. Western blotting assays
Total RNA of cells was extracted by using TRIzol (Invitrogen), and 1 μg RNA were reverse transcribed to cDNA by using reverse transcription kit (Takara, Dalian China). The qRT-PCR was performed using the SYBR-Green mix (Invitrogen) on a Roche LightCycler 480 system (Roche Diagnostics, Germany). GAPDH was used as an endogenous control. Relative expression of CAV1 was calculated using the power formula: 2-ΔCt (ΔCt = Ct CAV1–CtGAPDH). The CAV1 and GAPDH primer sequences were listed as following: CAV1 Forward: 5′-CATCCCGATGGCACTCATCTG-3′; CAV1 Reverse: 5′-TGCACTGAATCTCAATCAATCAGGAAG-3′;
Tissues or cells were lysed by using RIPA lysis buffer containing 1 mM Phenylmethylsulfonyl fluoride (PMSF) and a protease inhibitor cocktail tablet (Roche). The concentration of the protein was checked by BCA kit (Beyotime Institute of Biotechnology) according to manufacturer's protocols. 30 μg total proteins were separated by SDS-PAGE and then transferred onto PVDF membranes (Millipore, Bedford, MA, USA). After being blocked in 5% nonfat milk for 1 h, the membranes were incubated with primary antibody against CAV1 (Santa Cruz), Tubulin, β-actin and Flag (Beyotime Institute of Biotechnology) overnight at 4 ℃. The next day, the membranes were incubated for 2 h with 5
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Fig. 6. CAV1 expression is upregulated in sunitinib-resistant cells. (A-B) The mRNA expression and protein levels of CAV1 in sunitinib-resistant cells and parental cells. (C) Immunofluorescence analysis of CAV1 expression in sunitinib-resistant cells and parental cells. These experiments were repeated three times at least. The error bars represent mean ± SD of three independent experiments (****, P < 0.0001, ***, P < 0.001, compared with the corresponding parental cells).
ccRCC subgroups was performed using the Mann-Whitney test. The correlation between CAV1 mRNA expression and clinicopathological parameters of ccRCC patients was performed by Pearson's chisquare test. Receiver Operator characteristic (ROC) curve analysis was applied to determine the optimal cutoff point with respect to discriminating different ccRCC patient classifications. The Kaplan-Meier curve and log-rank test were performed to evaluate the correlation between the level of CAV1 expression and the disease-free survival rate. The data of each group were presented as mean ± SD. Statistical significance was set when the p value is less than 0.05.
Table 2 IC50 values of sunitinib in different ccRCC cell lines. IC50 (μM) Cells
Vector
CAV1
si-NC
si-CAV1
786-O ACHN 786-O-R ACHN-R
10.05 9.86 14.21 15.38
25.78 19.74 13.18 15.76
9.78 9.92 15.99 15.27
12.56 11.83 14.45 15.26
3. Results secondary antibody. Finally, the proteins were exposed and quantified using ChemiDoc-XRS+ (Bio-Rad, USA).
3.1. CAV1 is elevated and associated with various clinicopathological parameters in ccRCC
2.10. Bioinformatic analysis of CAV1 expression in ccRCC We assessed CAV1 mRNA expression in ccRCC cancer tissues and para-cancer tissues from publically available TCGA-KIRC database. CAV1 expression was higher in ccRCC tissues compared with paracancer tissues (Fig. 1A). We then analyzed the relationship between CAV1 expression and clinicopathological parameters in ccRCC (Table 1). The TCGA-KIRC database containing 533 cases revealed significantly higher CAV1 expression in male compared with female ccRCC patients (Fig. 1B). Elevated CAV1 expression also correlated significantly with higher T stage, pathological TNM stage, M stage, and Grade stage in ccRCC (Fig. 1C-F). However, the expression of cav1 was not associated with age, lymph node metastasis (Supplemental Fig S1). These data indicate that CAV1 expression is elevated and associated with various clinicopathological parameters in ccRCC.
The mRNA levels of CAV1 in ccRCC tissues and para-cancer tissues and clinicopathological data about age, gender, T stage, N stage, M stage, G stage, TNM stage, DFS of patients in TCGA_KIRC dataset were downloaded from the cBioPortal. 2.11. Statistical analysis The statistical analysis was performed using SPSS statistical software 22.0 (IBM SPSS, Chicago, IL, USA) or GraphPad Prism 6.0 (GraphPad software, Inc., USA). The statistical significance between the different sunitinib intervention groups was performed using Student's two-tailed t-test. The analysis of CAV1 mRNA expression in 6
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Fig. 7. The effects of CAV1 expression on sunitinib sensitivity in sunitinib-resistant and parental cells. (A, C) The effect of CAV1 overexpression on sunitinib sensitivity in sunitinibresistant cells and parental cells. (B, D) The effect of CAV1 knockdown on sunitinib sensitivity in sunitinib-resistant cells and parental cells. The error bars represent mean ± SD of three independent experiments (***, P < 0.001, **, P < 0.01, *, P < 0.05, compared with the corresponding control).
expression group (p < 0.0001). Moreover, we conducted disease-free survival analysis towards the expression of CAV1 mRNA in subgroups of ccRCC patients. Our results showed that high CAV1 expression could be a potential prognostic factor for ccRCC patients with T1 + T2 stage (Fig. 2B, p = 0.0072), T3 + T4 stage (Fig. 2C, p = 0.0307), N0 stage (Fig. 2D, p = 0.002), M0 stage (Fig. 2E, p = 0.0333), G3 + G4 stage (Fig. 2F, p = 0.0018), Male (Fig. 2G, p = 0.0010), Age ≤ 60 years (Fig. 2H, p = 0.022), Age > 60 years (Fig. 2I, p = 0.0003). However, high CAV1 expression exhibited no significant correlation with DFS of ccRCC patients with N1 stage, M1 stage, G1+G2 stage, female (Supplemental Fig S2).
3.2. The correlation between high CAV1 expression and poor diseasefree survival in ccRCC patients To investigate the prognostic value of CAV1, the correlation between CAV1 expression and disease-free survival time of ccRCC patients was analyzed by Kaplan-Meier, and the results were examined by the log-rank test (Fig. 2A-I). According to the median expression value of CAV1 mRNA level, the total 434 ccRCC patients from TCGAKIRC database were divided into low CAV1 expression group and high CAV1 expression group. As shown in Fig. 2A, high CAV1 expression group had poorer disease-free survival time (DFS) than low CAV1 7
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Fig. 8. The effects of CAV1 on migration and invasion in 786-O and ACHN cell lines in vitro. (A, B) The CAV1 protein levels were successfully knocked down or overexpressed in 786-O and ACHN cells. (C, D) The wound healing assays in CAV1-knockdown or CAV1-introduced 786-O and ACHN cells. (E, F) The transwell invasion assays in CAV1-knockdown or CAV1introduced 786-O and ACHN cells. The error bars represent mean ± SD of three independent experiments (**, P < 0.01, *, P < 0.05, compared with the corresponding parental cells).
3.5. Establishment of sunitinib-resistant cells in ccRCC cells
3.3. The association between high CAV1 expression and diagnostic value in ccRCC patients
Two sunitinib-resistant cell lines (786-O and ACHN) were established by continuous low-dose stimulation in conjunction with intermittent high-dose pulses of sunitinib as described and characterized [13]. These are designated as 786-O-R and ACHN-R cells, respectively. Then, we evaluated sensitivity of cells to sunitinib in sunitinib-resistant cells and their parental cells. As indicated in Fig. 5A-B, 786-O-R and ACHN-R showed higher cell viability compared with their parental cells after various concentrations of sunitinib administration. IC50 values of 786-O-R and ACHN-R were increased by more than 2-fold relative to that of their parental cells, respectively (Fig. 5C). These results showed that sunitinib-resistant cell lines were successfully constructed, and sunitinib-resistant cells had higher sunitinib tolerance.
To explore the diagnostic value of CAV1 in ccRCC, the ROC curves for the clinicopathological parameters were analyzed. ROC curve showed that CAV1 could effectively distinguish ccRCC from paracancer tissues yielding an area under the curve (AUC) of 0.9515 (95% CI: 0.9233–0.9796; p < 0.0001) with a sensitivity of 92.87% and a specificity of 91.67% when the cutoff value was 0.3973 (Fig. 3A). In addition, we also performed ROC curve analysis towards the expression of CAV1 mRNA in subgroups of ccRCC patients against T stage, N stage, M stage, G stage, TNM stage and DFS status. Our results indicated that high CAV1 expression could be a potential diagnostic indicators for ccRCC patients with TNM (I + II) / (III + IV) stage (Fig. 3B, AUC = 0.6089, p = 0.0001822), (T1 + T2)/(T3 + T4) stage (Fig. 3C, AUC = 0.5898, p = 0.002485), M0/M1 stage (Fig. 3D, (G1 + G2)/(G3 + G4) AUC = 0.6311, p = 0.002099), (G1 + G2)/(G3 + G4) stage (Fig. 3E, AUC = 0.5632, p = 0.02435), DFS status (Fig. 3F, AUC = 0.6702, p < 0.0001). However, high CAV1 expression could not differentiate ccRCC patients with N1 stage from ccRCC patients with N0 stage (data not shown).
3.6. CAV1 is overexpressed in sunitinib-resistant cell lines Previous studies demonstrated that CAV1 was involved in drug resistance and promoted cell migration and invasion in a variety of tumors. Based on previous research results, we first examined the levels of CAV1 expression in sunitinib-resistant cells and corresponding parental cells by qRT-PCR, WB and immunofluorescence. As shown in Fig. 6A-B, the sunitinib-resistant cells exhibited a significant higher mRNA expression and protein levels of CAV1 compared with the corresponding parental cells. To further confirm the high expression of CAV1 in sunitinib-resistant cell lines, immunofluorescence experiments were performed in sunitinib-resistant cells and parental cells. The results of immunofluorescence showed that the levels of CAV1 expression in 786-O-R and ACHN-R cells were significantly higher than that in 786-O and ACHN cells (Fig. 6C). Data above indicated that sunitinib-resistant cell lines had a higher CAV1 expression compared with corresponding parental cell lines.
3.4. High level of CAV1 expression is validated in ccRCC cells and tissues To further confirm the results of the TCGA database, CAV1 was subjected to western blot detection in ccRCC cells and tissues (Fig. 4A-B). IHC assay was also performed in 30 paired ccRCC tissues and para-cancer tissues (Fig. 4C). All results revealed that CAV1 protein level from ccRCC cells and tissues was significantly higher than immortalized renal epithelial cells and para-cancer tissues, respectively. 8
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growth, progression and metastasis in hepatocellular carcinoma [18]. On the contrary, CAV1 acts as a tumor suppressor gene in some malignancies, where low expression of CAV1 contributes to tumor progression. For instance, CAV1 is down-regulated and inhibits tumor growth in alveolar rhabdomyosarcoma [19]. As a tumor suppressor gene, CAV1 is down-regulated at both mRNA and protein levels in vestibular schwannomas [20]. In addition, the loss of stromal CAV1 is associated with metastasis and poor survival in metastatic malignant melanoma [21]. The results of these studies indicate that CAV1 may play a different role in different malignancies. Different gene function of CAV1 in different tumors may be attributed to tumor histological differences and tumor microenvironments differences. Numerous studies have reported that CAV1 is involved in drug resistance in tumors targeted therapy [5,6,22–24]. Mutation of CAV1 in tumor cells increases the transport activity of P-glycoprotein and reduces antitumor drug killing ability in non-small cell lung cancer cells [25]. CAV1 promotes apoptosis resistance induced by chemotherapy in Ewing's sarcoma by regulating PKCα phosphorylation [6]. CAV1 is up-regulated by chemotherapy and increases resistance to epirubicin in gastric cancer cells [5]. The knockdown of CAV1 lowers the transport activity of ABCG2 and increases the sensitivity of breast cancer to chemotherapy drugs mitoxantrone [22]. CAV1 influences drug resistance in esophageal squamous cell carcinoma by regulating the expression of MRP1 and P-glycoprotein [23]. CAV1 promote chemotherapy resistance in breast cancer stem cells by regulating beta-catenin/ABCG2 signaling pathway [26]. In the study of renal cell carcinoma, previous reports have indicated that CAV1 is highly expressed in renal cell carcinoma and associated with poor prognosis and metastasis [3,27–32], which are consistent with our findings. However, the roles of CAV1 in sunitinib resistance of renal cell carcinoma remain unclear. As for the underlying mechanism of overexpression of CAV1, it has been reported that miR-124 can reduce CAV1 expression by targeting CAV1 mRNA [33]. Therefore, we hypothesized that CAV1 overexpression may be associated with decreased expression of miR-124 in renal cell carcinoma. As for the effector of CAV1 downstream, CAV1 can activate AKT through Interactions with and Inhibition of PP1 and PP2A in prostate cancer cells [34]. In addition, previous report indicates that PP2A is down-regulated in kidney cancer [35]. These reports suggest that PP2A may be a potential downstream target of CAV1. CAV1 can also interact with AR to promote AR transcriptional activity [36]. We will further explore the potential mechanisms of CAV1 overexpression and its effectors in kidney cancer in future studies. In our study, we found that CAV1 expression was elevated in kidney cancer cells and tissues, consistent with many previous studies in other tumor types. Moreover, we discovered that high CAV1 expression was associated with poor DFS and could serve as diagnostic indicator in ccRCC patients with different clinicopathological stages. We hypothesized that elevated CAV1 may play an important role in the development and progression of renal cell carcinoma. In order to verify our speculation, we had done a variety of functional experiments in renal carcinoma cell lines. The results of functional experiments show that CAV1 promotes the migration and invasion of renal cell carcinoma, indicating that CAV1 may affect the biological behavior of renal cell carcinoma. Due to the existence of intrinsic or acquired drug resistance mechanisms, resistance to first-line drug sunitinib still remains a major problem to be solved in clinical therapy, and CAV1 is reported to be involved in drug resistance in a variety of tumors. Unfortunately, there is no study on the role of CAV1 in sunitinib resistance mechanisms in renal cancer. We established sunitinib-resistant cell lines in kidney cancer cells with continuous low-dose stimulation in conjunction with intermittent high-dose pulses of sunitinib. The expression of CAV1 in sunitinib-resistant cell lines was significantly higher than that in parental cell lines. Plasmid-mediated overexpression of CAV1 significantly increased resistance to sunitinib in parental and drug resistant cell lines, while knockdown of CAV1 expression did not increase the sensitivity of cells to sunitinib. Due to inconsistent with our expected
3.7. The effects of CAV1 knockdown or overexpression on sunitinib sensitivity and cells viability To investigate the role of CAV1 in sunitinib resistance procedures, we successfully overexpressed or knocked down CAV1 in sunitinibresistant cell lines and parental cell lines. We then examined the cells viability in sunitinib-resistant and parental cell lines at various sunitinib concentrations after CAV1 being overexpressed or knocked down, and the IC50 values of different intervention groups cells were listed in Table 2. As shown in Fig. 7A+C, the cell lines of CAV1 overexpression possessed a higher cell viability compared with the corresponding control cell lines. Contrary to our desired outcome, CAV1 knockdown did not enhance sunitinib sensitivity in ccRCC cells (Fig. 7B, D). At some different sunitinib concentrations, CAV1 knockdown cells even revealed a clearly more sunitinib-resistant phenotype rather than the desired sunitinib-sensitive phenotype. We speculated that the reduced cells proliferation rate of CAV1 knockdown cells resulted in insensitivity of these cells to sunitinib and other drugresistant genes might continue to maintain cell-resistant phenotype in the case of CAV1 knockdown. These results suggested that CAV1 overexpression might contribute to cellular tolerability of sunitinib. 3.8. CAV1 promotes the migration and invasion of ccRCC cells in vitro To determine how CAV1 contributes to aggressiveness of ccRCC, we first examined whether CAV1 increased cell migration and invasion in vitro, the critical features of tumor metastatic phenotype. Given the high CAV1 expression in all ccRCC cell lines detected, we first selected a CAV1 si-RNA knockdown method. Two CAV1 si-RNA oligonucleotide sequences were used, resulting in consistent CAV1 knockdown (Fig. 8A). Scratches of 786-O and ACHN cells healed more rapidly than corresponding CAV1 knockdown cells in wound-healing assays (Fig. 8C). 786-O and ACHN cells also possessed a faster invasion capacity than corresponding CAV1 knockdown cells in transwell assays (Fig. 8E). Next, we transferred the CAV1 plasmid to 786-O and ACHN cells, resulting in overexpression of CAV1 (Fig. 8B). Scratch assays and transwell invasion experiments demonstrated that cells migration and invasion of CAV1-expressing cells were faster compared with the corresponding control cell lines (Figs. 8D and F). These data demonstrated that CAV1 could promote the migration and invasion capacity of ccRCC cells. 4. Discussion Existing understanding about the molecular mechanisms underlying disease progression and sunitinib resistance in ccRCC is still incomplete. Cancer metastasis remains the major cause of death in ccRCC patients. Receptor tyrosine kinase inhibitors (RTKI), including sunitinib, are currently the first-line therapeutic agents for metastatic and advanced renal cell carcinoma. However, patients who are completely or continuously sensitive to RTKI are very rare, and even those who are initially sensitive to RTKI will subsequently develop and progress into RTKI resistance after an average of 6–15 months of treatment [14]. Thus, cancer metastasis and drug resistance remain the major clinical problems in ccRCC. However, the specific molecular mechanisms of cancer metastasis and drug resistance are still not very clear in ccRCC. Cumulative evidence suggests that CAV1 is significantly overexpressed in a variety of human malignant cancers, including cancers of liver [4], bladder [9], prostate [7], brain [15], suggesting that it may play an important role in the initiation and progression of these tumors. Previous study has confirmed that overexpression of CAV1 is associated with the recurrence of progressive prostate cancer [16]. CAV1 upregulation is associated with poor prognosis and enhanced metastatic capacity in lung cancer [17]. CAV1 contributes to tumor 9
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results, we hypothesized that other drug-resistant genes might continue to maintain cell-resistant phenotype in the case of CAV1 knockdown. However, there is a drawback in our study that the role of CAV1 in the animal model of kidney cancer and the mechanism of sunitinib resistance in vivo remain unclear. In future studies, we will explore the biological effects of CAV1 and the mechanism of sunitinib resistance in vivo, aiming to elucidate the role of CAV1 in the progression of renal cell carcinoma and sunitinib resistance. In conclusion, our study confirms that CAV1 is elevated in RCC tissues and cell lines as well as in sunitinib-resistant cell lines and high CAV1 expression can serve as a useful molecular marker for the diagnosis and prognosis of ccRCC patients. Moreover, our findings confirm that CAV1 promotes the migration and invasion of renal carcinoma cells and increases the tolerance of renal carcinoma cells to sunitinib. Although the in vivo function of CAV1 needs to be evaluated, the data derived from our experiments strongly support the idea that the development of CAV1-targeting drug may increase the efficiency of ccRCC treatment and improve the therapy outcome for ccRCC patients, especially those presenting with advanced and metastatic ccRCC. Statement of conflict of interest The authors state that they have no conflicts of interest. Acknowledgements This research was supported by the National Natural Science Foundation of China (Grant No. 81672528, 81672524 & 81272560), the Program for New Century Excellent Talents in University from the Department of Education of China (NCET-08-0223), the National High Technology Research and Development Program of China (863 Program) (2012AA021101). Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at doi:10.1016/j.yexcr.2017.07.004. References [1] R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics, 2017, CA: Cancer J. Clin. 67 (1) (2017) 7–30 (PubMed PMID: 28055103). [2] S.F. Tavazoie, C. Alarcon, T. Oskarsson, D. Padua, Q. Wang, P.D. Bos, et al., Endogenous human microRNAs that suppress breast cancer metastasis, Nature 451 (7175) (2008) 147–152 (PubMed PMID: 18185580. Pubmed Central PMCID: 2782491). [3] I. Tamaskar, T.K. Choueiri, L. Sercia, B. Rini, R. Bukowski, M. Zhou, Differential expression of caveolin-1 in renal neoplasms, Cancer 110 (4) (2007) 776–782 (PubMed PMID: 17594718). [4] E.Y. Tse, F.C. Ko, E.K. Tung, L.K. Chan, T.K. Lee, E.S. Ngan, et al., Caveolin-1 overexpression is associated with hepatocellular carcinoma tumourigenesis and metastasis, J. Pathol. 226 (4) (2012) 645–653 (PubMed PMID: 22072235). [5] G. Yuan, I. Regel, F. Lian, T. Friedrich, I. Hitkova, R.D. Hofheinz, et al., WNT6 is a novel target gene of caveolin-1 promoting chemoresistance to epirubicin in human gastric cancer cells, Oncogene 32 (3) (2013) 375–387 (PubMed PMID: 22370641). [6] O.M. Tirado, C.M. MacCarthy, N. Fatima, J. Villar, S. Mateo-Lozano, V. Notario, Caveolin-1 promotes resistance to chemotherapy-induced apoptosis in Ewing's sarcoma cells by modulating PKCalpha phosphorylation, Int. J. Cancer 126 (2) (2010) 426–436 (PubMed PMID: 19609943. Pubmed Central PMCID: 2794946). [7] G. Ayala, M. Morello, A. Frolov, S. You, R. Li, F. Rosati, et al., Loss of caveolin-1 in prostate cancer stroma correlates with reduced relapse-free survival and is functionally relevant to tumour progression, J. Pathol. 231 (1) (2013) 77–87 (PubMed PMID: 23729330. Pubmed Central PMCID: 3978784). [8] C.C. Ho, S.H. Kuo, P.H. Huang, H.Y. Huang, C.H. Yang, P.C. Yang, Caveolin-1 expression is significantly associated with drug resistance and poor prognosis in advanced non-small cell lung cancer patients treated with gemcitabine-based chemotherapy, Lung Cancer 59 (1) (2008) 105–110 (PubMed PMID: 17850918). [9] T. Qayyum, G. Fyffe, M. Duncan, P.A. McArdle, M. Hilmy, C. Orange, et al., The interrelationships between Src, Cav-1 and RhoGD12 in transitional cell carcinoma of the bladder, Br. J. Cancer 106 (6) (2012) 1187–1195 (PubMed PMID: 22353809. Pubmed Central PMCID: 3304420). [10] L. Liu, H.X. Xu, W.Q. Wang, C.T. Wu, T. Chen, Y. Qin, et al., Cavin-1 is essential for the tumor-promoting effect of caveolin-1 and enhances its prognostic potency in pancreatic cancer, Oncogene 33 (21) (2014) 2728–2736 (PubMed PMID: 23770857).
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Enhanced expression of caveolin-1 possesses diagnostic and prognostic value and promotes cell migration, invasion and sunitinib resistance in the clear cell renal cell carcinoma HaiLong Ruana,1, Xiang Lia,1, HongMei Yangb, ZhengShuai Songa, JunWei Tonga, Qi Caoa, KeShan Wanga, Wen Xiaoa, HaiBin Xiaoa, XuanYu Chena,c,d, GuangHua Xua, Lin Baoa, ZhiYong Xionga, ChangFei Yuana, Lei Liua, Yan Qub, WenJun Hub, YaoYing Gaob, ZeYuan Rub, ⁎ Ke Chena, XiaoPing Zhanga, a Department of Urology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1277 Jiefang Avenue, Wuhan 430022, Hubei Province, China b Department of Pathogenic Biology, School of Basic Medicine, Huazhong University of Science and Technology, 13 Hangkong Road, Wuhan 430030, Hubei Province, China c Department of Urology, Zhejiang Provincial People's Hospital, Hangzhou 310014, China d Department of Neuroscience and Regenerative Medicine, Medical College of Georgia, Augusta University, Augusta, GA 30912, USA
A R T I C L E I N F O
A BS T RAC T
Keywords: CAV1 Sunitinib-resistance Invasion Prognosis Diagnosis ccRCC
Caveolin-1 (CAV1) has been identified to be up-regulated in many cancers, including clear cell renal cell carcinoma (ccRCC). However, its potential function is still unclear in ccRCC. In this study, we demonstrated that CAV1 was frequently overexpressed in renal cell carcinoma tissues and cells, and was significantly associated with various clinicopathological parameters. In addition, high CAV1 expression was associated with poor disease-free survival (DFS) rate and could serve as a useful diagnostic indicator in ccRCC patients with different clinicopathological stages. Functional experiments demonstrated that CAV1 knockdown inhibited cell migration and invasion, whereas overexpression of CAV1 promoted cell migration and invasion in ccRCC. Moreover, CAV1 expression was up-regulated in sunitinib-resistant renal cancer cell lines, and its overexpression promoted sunitinib resistance. In general, our results confirm that CAV1 plays an important role in the metastasis of kidney cancer and induces sunitinib resistance, so CAV1 function suppression may become a promising clinical treatment strategy during renal cell carcinoma metastasis and sunitinib resistance.
1. Introduction Renal cell carcinoma (RCC) is the most common malignant neoplasm in the kidney, which accounts for approximately 80–90% renal malignancies and about 2–3% adult malignancies. It is predicted that approximately 63,990 new patients of renal cancer and 14,400 deaths from renal cancer will emerge in the U.S in 2017 [1]. RCC comprises a variety of different pathologic subtypes. Clear cell RCC (ccRCC) is the most common subtype that is generally accompanied by high rate of metastasis and mortality, and nonsensitive to conventional chemotherapy and radiotherapy. Tumor metastasis is the leading cause of death in cancer-bearing patients [2]. Prompt surgical treatment for localized RCC patients can achieve a good prognosis and survival rate. Disappointingly, approximately 30% of patients with RCC have pro⁎
1
gressed into local invasion or distant metastasis when clinically diagnosed, leading to poor prognosis. In recent years, receptor tyrosine kinase inhibitors (RTKI), including sunitinib, have become first-line therapeutic agents for metastatic and advanced RCC. However, due to intrinsic or acquired resistance to RTKI and high side effects of drugs, most RCC patients are restricted to use these drugs. Therefore, it is urgently needed to explore the metastatic and RTKI-resistant mechanisms in RCC and develop new therapeutic drugs. Caveolins are structurally and functionally evolutionarily conserved and represent the major components of caveolae, which are specialized lipid raft microdomains of the cytoplasmic membrane involved in intracellular signal transduction pathways that regulate cell growth, adhesion and survival [3]. Caveolin-1 (CAV1), belonging to the caveolin supergene family, is originally identified as a 24 kDa protein that form
Corresponding author. E-mail address: [email protected] (X. Zhang). These authors contribute equally to this study.
http://dx.doi.org/10.1016/j.yexcr.2017.07.004 Received 11 June 2017; Received in revised form 29 June 2017; Accepted 1 July 2017 0014-4827/ © 2017 Published by Elsevier Inc.
Please cite this article as: Ruan, H., Experimental Cell Research (2017), http://dx.doi.org/10.1016/j.yexcr.2017.07.004
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Fig. 1. The level of CAV1 is upregulated and correlated with various clinicopathological parameters in ccRCC tissues. (A-F) The mRNA level of CAV1 in ccRCC was downloaded from the TCGA_KIRC dataset containing 72 para-cancer tissues and 533 ccRCC tissues. The mRNA levels of CAV1 were compared in different clinicopathological parameters: (A) cancer versus para-cancer, (B) gender, (C) T stage, (D) TNM stage, (E) M stage, (F) G stage.
oncogenic and drug-resistant gene in RCC tumor microenvironment and provided a firm theoretical principle for consideration of CAV1 combining with receptor tyrosine kinase inhibitors as a new therapy for metastatic and advanced RCC.
caveolae at the plasma membrane. Dysregulation of CAV1 expression is often associated with tumorigenesis, progression, prognosis, metastasis, multidrug- resistance [4–8]. CAV1 was upregulated in prostate cancer [7], bladder cancer [9], pancreas cancer [10]. On the other hand, CAV1 was downregulated in colon cancer [11], breast cancer [12]. Thus, the biological role of CAV1 in tumor cells is quite complicated depending on the origin and the microenvironment of the tumor. However, few studies have been carried out to investigate correlation between CAV1 expression and RTKI-resistance in renal cell carcinoma and the biological behavior of CAV1 in renal cancer cells. In our research, we for the first time demonstrated that CAV1 is significantly upregulated in sunitinib-resistance RCC cells and promotes sunitinib resistance in RCC cells. Moreover, we identified CAV1 was overexpressed in RCC cells and tissues, and promoted cell migration and invasion. These data indicated that CAV1 might be an
2. Materials and methods 2.1. Renal cancer tissue samples We collected 60 pairs of ccRCC and adjacent normal renal tissues from nephrectomies operated at the Wuhan Union Hospital between 2015 and 2016. Resected tissues were divided into two halves: one half was promptly frozen in liquid nitrogen for subsequent western blot experiments; the other half was fixed with formalin and paraffinembedded for matched immunohistochemistry assays. Before surgery, 2
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all patients did not receive any chemotherapy or radiotherapy. All surgical patients had signed an informed consent form. The study and experimental procedures were approved by the Human Research Ethics Committee of Huazhong University of Science and Technology.
Table 1 Correlation between CAV1 mRNA expression and clinicopathological parameters of ccRCC patients. CAV1 mRNA expression Parameter
Age (years) Gender T stage N stage M stage G stage TNM stage
< = 60 > 60 Male Female T1 + T2 T3 + T4 N0 + NX N1 M0 + MX M1 G1 + G2 + GX G3 + G4 I + II III + IV
Number
Low (n = 263)
High (n = 263)
261 265 341 185 337 189 510 16 448 78 247 279 319 207
128 135 147 116 179 84 257 6 238 25 136 127 174 89
133 130 194 69 158 105 253 10 210 53 111 152 145 118
P value
2.2. Cell culture and drug intervention The human renal cancer cell lines 786-O, ACHN, A498, Caki-1, OSRC-2 and renal tubular epithelial cell line HK-2 were purchased from The American Type Culture Collection (ATCC, USA). Cells were maintained in DMEM high glucose medium (Google Biotechnology Co., Ltd, Wuhan, China) containing 10% FBS (Lilac Garden Technology Co., Ltd, Wuhan, China) and 1% penicillin-streptomycin at 37 ℃ in a 5% CO2 incubator. For drug intervention, sunitinib (Pfizer) was dissolved in DMSO at a concentration of 15 mg/ml and then added to the culture medium of renal cancer cells at the indicated concentrations.
0.663 0.000 0.056 0.310 0.001 0.029
2.3. Establishment of sunitinib-resistant cell lines 0.010
To establish sunitinib-resistant renal carcinoma cell lines, 786-O and ACHN were established by continuous low-dose stimulation in
Fig. 2. High level of CAV1 mRNA predicts poor disease-free survival rate in ccRCC patients. (A) The ccRCC patients from TCGA-KIRC database were divided into low CAV1 expression group and high CAV1 expression group according to the median expression value of CAV1 mRNA level. The correlation between CAV1 expression and disease-free survival time of total ccRCC patients was analyzed by Kaplan-Meier. (B-I) Disease-free survival analysis towards the expression of CAV1 mRNA was performed in subgroups of ccRCC patients: (B) T1 + T2 stage, (C) T3 + T4 stage, (D) N0 stage, (E) M0 stage, (F) G3 + G4 stage, (G) Male, (H) Age ≤ 60 years, (I) Age > 60 years.
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Fig. 3. High CAV1 expression serves as a diagnostic indicator in ccRCC patients. (A) ROC curve showed that CAV1 could effectively distinguish ccRCC from para-cancer tissues. The AUC was 0.9515 (p < 0.0001). (B-F) ROC curve analysis towards the expression of CAV1 mRNA in subgroups of ccRCC patients against TNM stage (B), T stage (C), M stage (D), G stage (E) and DFS status (F).
2.5. Immunohistochemistry assay
conjunction with intermittent high-dose pulses of sunitinib as described and characterized [13]. The IC50 values of the sunitinibresistant cell lines are more than twice that of the parental cell lines.
Briefly, ccRCC tissues and corresponding para-cancer tissues were formalin-fixed, dehydrated, and paraffin-embedded. The tissue sections were incubated with rabbit CAV1 polyclonal antibody (1:100, Santa Cruz, CA, USA) overnight at 4 ℃. The next day, tissue sections were washed three times with PBS and then incubated with horseradish peroxidase conjugated secondary antibody.
2.4. Transient transfection assay The si-RNA oligonucleotide sequences targeting CAV1 (si-CAV1) and si-RNA Negative Control (si-NC) were synthesized by Gene-Pharma (ShangHai, China). BLAST alignment of all si-RNA sequences was performed to ensure that they had no off-target effect. The si-CAV1 and si-NC with a final concentration of 50 nM were transfected with lipofectamine 2000 (Invitrogen, USA) according to the manufacturer's protocols. The CAV1 plasmid (CAV1) and Negative Control vector (Vector) were purchased from Vigene Biosciences (Shan Dong, China). A total 2 μg of CAV1 or Vector plasmid were transfected with lipofectamine 2000 according to the manufacturer's recommendations. Cells were collected for subsequent assays at 48 h after transfection. The si-CAV1 sequences were shown as following: #1: 5′-CGACGTGGTCAAGATTGACTT-3′; #2: 5′GCTTCCTGATTGAGATTCAGT-3′;
2.6. Immunofluorescence The appropriate numbers of cells were planted on glass coverslips, washed three times with PBS, fixed in 4% paraformaldehyde for 10 min. Cells were blocked for 1 h with 3% BSA and then were incubated with CAV1 primary antibodies overnight at 4 ℃. The next day, these cells were incubated for 1 h with CY3-conjugated secondary antibodies, and then stained with DAPI. Finally, these cells were photographed under fluorescence microscopy.
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Fig. 4. CAV1 expression is elevated in our ccRCC cells and tissues. (A-B) Western blot analysis of CAV1 expression in renal cancer cell lines and ccRCC tissues. (C) Immunohistochemistry (IHC) analysis of CAV1 expression in ccRCC tissues and paracancer tissues. Representative images were shown.
2.7. Wound healing assays and transwell invasion assays For wound healing assays, cells transfected with si-CAV1 and si-NC were seeded in 6-wells plate. After reaching 80–90% confluent, the cells were scratched with 10-μl pipette tips, washed, and then photographed at 0 h and 24 h. In vitro cell invasion assays were carried out using 24-well transwell chambers with 8.0 µm pore size (Corning, NY, USA). For the invasion assay, 2 × 104 (786-O, ACHN) cells transfected with si-CAV1 and si-NC were added to the upper chamber of the transwell chamber precoated with Matrigel (BD Biosciences, NJ, USA). The medium containing 10% FBS was added to the lower chamber. After 24 h incubation, invaded cells were fixed with methanol and then stained with 0.05% crystal violet. The invaded cells were counted in 5 random fields.
Fig. 5. The sunitinib sensitivity curve of sunitinib-resistant and parental cells. (A) The sunitinib sensitivity curve of 786-O and 786O-R. (B) The sunitinib sensitivity curve of ACHN and ACHN-R. (C) IC50 value of sunitinib-resistant and parental cells. The error bars represent mean ± SD of three independent experiments (**, P < 0.01, *, P < 0.05, compared with the corresponding parental cells).
GAPDH Forward: 5′-GGTGAAGGTCGGAGTCAACGG-3′; GAPDH Reverse: 5′-GAGGTCAATGAAGGGGTCATTG-3′.
2.8. Quantitative real-time PCR assays (qRT-PCR)
2.9. Western blotting assays
Total RNA of cells was extracted by using TRIzol (Invitrogen), and 1 μg RNA were reverse transcribed to cDNA by using reverse transcription kit (Takara, Dalian China). The qRT-PCR was performed using the SYBR-Green mix (Invitrogen) on a Roche LightCycler 480 system (Roche Diagnostics, Germany). GAPDH was used as an endogenous control. Relative expression of CAV1 was calculated using the power formula: 2-ΔCt (ΔCt = Ct CAV1–CtGAPDH). The CAV1 and GAPDH primer sequences were listed as following: CAV1 Forward: 5′-CATCCCGATGGCACTCATCTG-3′; CAV1 Reverse: 5′-TGCACTGAATCTCAATCAATCAGGAAG-3′;
Tissues or cells were lysed by using RIPA lysis buffer containing 1 mM Phenylmethylsulfonyl fluoride (PMSF) and a protease inhibitor cocktail tablet (Roche). The concentration of the protein was checked by BCA kit (Beyotime Institute of Biotechnology) according to manufacturer's protocols. 30 μg total proteins were separated by SDS-PAGE and then transferred onto PVDF membranes (Millipore, Bedford, MA, USA). After being blocked in 5% nonfat milk for 1 h, the membranes were incubated with primary antibody against CAV1 (Santa Cruz), Tubulin, β-actin and Flag (Beyotime Institute of Biotechnology) overnight at 4 ℃. The next day, the membranes were incubated for 2 h with 5
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Fig. 6. CAV1 expression is upregulated in sunitinib-resistant cells. (A-B) The mRNA expression and protein levels of CAV1 in sunitinib-resistant cells and parental cells. (C) Immunofluorescence analysis of CAV1 expression in sunitinib-resistant cells and parental cells. These experiments were repeated three times at least. The error bars represent mean ± SD of three independent experiments (****, P < 0.0001, ***, P < 0.001, compared with the corresponding parental cells).
ccRCC subgroups was performed using the Mann-Whitney test. The correlation between CAV1 mRNA expression and clinicopathological parameters of ccRCC patients was performed by Pearson's chisquare test. Receiver Operator characteristic (ROC) curve analysis was applied to determine the optimal cutoff point with respect to discriminating different ccRCC patient classifications. The Kaplan-Meier curve and log-rank test were performed to evaluate the correlation between the level of CAV1 expression and the disease-free survival rate. The data of each group were presented as mean ± SD. Statistical significance was set when the p value is less than 0.05.
Table 2 IC50 values of sunitinib in different ccRCC cell lines. IC50 (μM) Cells
Vector
CAV1
si-NC
si-CAV1
786-O ACHN 786-O-R ACHN-R
10.05 9.86 14.21 15.38
25.78 19.74 13.18 15.76
9.78 9.92 15.99 15.27
12.56 11.83 14.45 15.26
3. Results secondary antibody. Finally, the proteins were exposed and quantified using ChemiDoc-XRS+ (Bio-Rad, USA).
3.1. CAV1 is elevated and associated with various clinicopathological parameters in ccRCC
2.10. Bioinformatic analysis of CAV1 expression in ccRCC We assessed CAV1 mRNA expression in ccRCC cancer tissues and para-cancer tissues from publically available TCGA-KIRC database. CAV1 expression was higher in ccRCC tissues compared with paracancer tissues (Fig. 1A). We then analyzed the relationship between CAV1 expression and clinicopathological parameters in ccRCC (Table 1). The TCGA-KIRC database containing 533 cases revealed significantly higher CAV1 expression in male compared with female ccRCC patients (Fig. 1B). Elevated CAV1 expression also correlated significantly with higher T stage, pathological TNM stage, M stage, and Grade stage in ccRCC (Fig. 1C-F). However, the expression of cav1 was not associated with age, lymph node metastasis (Supplemental Fig S1). These data indicate that CAV1 expression is elevated and associated with various clinicopathological parameters in ccRCC.
The mRNA levels of CAV1 in ccRCC tissues and para-cancer tissues and clinicopathological data about age, gender, T stage, N stage, M stage, G stage, TNM stage, DFS of patients in TCGA_KIRC dataset were downloaded from the cBioPortal. 2.11. Statistical analysis The statistical analysis was performed using SPSS statistical software 22.0 (IBM SPSS, Chicago, IL, USA) or GraphPad Prism 6.0 (GraphPad software, Inc., USA). The statistical significance between the different sunitinib intervention groups was performed using Student's two-tailed t-test. The analysis of CAV1 mRNA expression in 6
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Fig. 7. The effects of CAV1 expression on sunitinib sensitivity in sunitinib-resistant and parental cells. (A, C) The effect of CAV1 overexpression on sunitinib sensitivity in sunitinibresistant cells and parental cells. (B, D) The effect of CAV1 knockdown on sunitinib sensitivity in sunitinib-resistant cells and parental cells. The error bars represent mean ± SD of three independent experiments (***, P < 0.001, **, P < 0.01, *, P < 0.05, compared with the corresponding control).
expression group (p < 0.0001). Moreover, we conducted disease-free survival analysis towards the expression of CAV1 mRNA in subgroups of ccRCC patients. Our results showed that high CAV1 expression could be a potential prognostic factor for ccRCC patients with T1 + T2 stage (Fig. 2B, p = 0.0072), T3 + T4 stage (Fig. 2C, p = 0.0307), N0 stage (Fig. 2D, p = 0.002), M0 stage (Fig. 2E, p = 0.0333), G3 + G4 stage (Fig. 2F, p = 0.0018), Male (Fig. 2G, p = 0.0010), Age ≤ 60 years (Fig. 2H, p = 0.022), Age > 60 years (Fig. 2I, p = 0.0003). However, high CAV1 expression exhibited no significant correlation with DFS of ccRCC patients with N1 stage, M1 stage, G1+G2 stage, female (Supplemental Fig S2).
3.2. The correlation between high CAV1 expression and poor diseasefree survival in ccRCC patients To investigate the prognostic value of CAV1, the correlation between CAV1 expression and disease-free survival time of ccRCC patients was analyzed by Kaplan-Meier, and the results were examined by the log-rank test (Fig. 2A-I). According to the median expression value of CAV1 mRNA level, the total 434 ccRCC patients from TCGAKIRC database were divided into low CAV1 expression group and high CAV1 expression group. As shown in Fig. 2A, high CAV1 expression group had poorer disease-free survival time (DFS) than low CAV1 7
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Fig. 8. The effects of CAV1 on migration and invasion in 786-O and ACHN cell lines in vitro. (A, B) The CAV1 protein levels were successfully knocked down or overexpressed in 786-O and ACHN cells. (C, D) The wound healing assays in CAV1-knockdown or CAV1-introduced 786-O and ACHN cells. (E, F) The transwell invasion assays in CAV1-knockdown or CAV1introduced 786-O and ACHN cells. The error bars represent mean ± SD of three independent experiments (**, P < 0.01, *, P < 0.05, compared with the corresponding parental cells).
3.5. Establishment of sunitinib-resistant cells in ccRCC cells
3.3. The association between high CAV1 expression and diagnostic value in ccRCC patients
Two sunitinib-resistant cell lines (786-O and ACHN) were established by continuous low-dose stimulation in conjunction with intermittent high-dose pulses of sunitinib as described and characterized [13]. These are designated as 786-O-R and ACHN-R cells, respectively. Then, we evaluated sensitivity of cells to sunitinib in sunitinib-resistant cells and their parental cells. As indicated in Fig. 5A-B, 786-O-R and ACHN-R showed higher cell viability compared with their parental cells after various concentrations of sunitinib administration. IC50 values of 786-O-R and ACHN-R were increased by more than 2-fold relative to that of their parental cells, respectively (Fig. 5C). These results showed that sunitinib-resistant cell lines were successfully constructed, and sunitinib-resistant cells had higher sunitinib tolerance.
To explore the diagnostic value of CAV1 in ccRCC, the ROC curves for the clinicopathological parameters were analyzed. ROC curve showed that CAV1 could effectively distinguish ccRCC from paracancer tissues yielding an area under the curve (AUC) of 0.9515 (95% CI: 0.9233–0.9796; p < 0.0001) with a sensitivity of 92.87% and a specificity of 91.67% when the cutoff value was 0.3973 (Fig. 3A). In addition, we also performed ROC curve analysis towards the expression of CAV1 mRNA in subgroups of ccRCC patients against T stage, N stage, M stage, G stage, TNM stage and DFS status. Our results indicated that high CAV1 expression could be a potential diagnostic indicators for ccRCC patients with TNM (I + II) / (III + IV) stage (Fig. 3B, AUC = 0.6089, p = 0.0001822), (T1 + T2)/(T3 + T4) stage (Fig. 3C, AUC = 0.5898, p = 0.002485), M0/M1 stage (Fig. 3D, (G1 + G2)/(G3 + G4) AUC = 0.6311, p = 0.002099), (G1 + G2)/(G3 + G4) stage (Fig. 3E, AUC = 0.5632, p = 0.02435), DFS status (Fig. 3F, AUC = 0.6702, p < 0.0001). However, high CAV1 expression could not differentiate ccRCC patients with N1 stage from ccRCC patients with N0 stage (data not shown).
3.6. CAV1 is overexpressed in sunitinib-resistant cell lines Previous studies demonstrated that CAV1 was involved in drug resistance and promoted cell migration and invasion in a variety of tumors. Based on previous research results, we first examined the levels of CAV1 expression in sunitinib-resistant cells and corresponding parental cells by qRT-PCR, WB and immunofluorescence. As shown in Fig. 6A-B, the sunitinib-resistant cells exhibited a significant higher mRNA expression and protein levels of CAV1 compared with the corresponding parental cells. To further confirm the high expression of CAV1 in sunitinib-resistant cell lines, immunofluorescence experiments were performed in sunitinib-resistant cells and parental cells. The results of immunofluorescence showed that the levels of CAV1 expression in 786-O-R and ACHN-R cells were significantly higher than that in 786-O and ACHN cells (Fig. 6C). Data above indicated that sunitinib-resistant cell lines had a higher CAV1 expression compared with corresponding parental cell lines.
3.4. High level of CAV1 expression is validated in ccRCC cells and tissues To further confirm the results of the TCGA database, CAV1 was subjected to western blot detection in ccRCC cells and tissues (Fig. 4A-B). IHC assay was also performed in 30 paired ccRCC tissues and para-cancer tissues (Fig. 4C). All results revealed that CAV1 protein level from ccRCC cells and tissues was significantly higher than immortalized renal epithelial cells and para-cancer tissues, respectively. 8
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growth, progression and metastasis in hepatocellular carcinoma [18]. On the contrary, CAV1 acts as a tumor suppressor gene in some malignancies, where low expression of CAV1 contributes to tumor progression. For instance, CAV1 is down-regulated and inhibits tumor growth in alveolar rhabdomyosarcoma [19]. As a tumor suppressor gene, CAV1 is down-regulated at both mRNA and protein levels in vestibular schwannomas [20]. In addition, the loss of stromal CAV1 is associated with metastasis and poor survival in metastatic malignant melanoma [21]. The results of these studies indicate that CAV1 may play a different role in different malignancies. Different gene function of CAV1 in different tumors may be attributed to tumor histological differences and tumor microenvironments differences. Numerous studies have reported that CAV1 is involved in drug resistance in tumors targeted therapy [5,6,22–24]. Mutation of CAV1 in tumor cells increases the transport activity of P-glycoprotein and reduces antitumor drug killing ability in non-small cell lung cancer cells [25]. CAV1 promotes apoptosis resistance induced by chemotherapy in Ewing's sarcoma by regulating PKCα phosphorylation [6]. CAV1 is up-regulated by chemotherapy and increases resistance to epirubicin in gastric cancer cells [5]. The knockdown of CAV1 lowers the transport activity of ABCG2 and increases the sensitivity of breast cancer to chemotherapy drugs mitoxantrone [22]. CAV1 influences drug resistance in esophageal squamous cell carcinoma by regulating the expression of MRP1 and P-glycoprotein [23]. CAV1 promote chemotherapy resistance in breast cancer stem cells by regulating beta-catenin/ABCG2 signaling pathway [26]. In the study of renal cell carcinoma, previous reports have indicated that CAV1 is highly expressed in renal cell carcinoma and associated with poor prognosis and metastasis [3,27–32], which are consistent with our findings. However, the roles of CAV1 in sunitinib resistance of renal cell carcinoma remain unclear. As for the underlying mechanism of overexpression of CAV1, it has been reported that miR-124 can reduce CAV1 expression by targeting CAV1 mRNA [33]. Therefore, we hypothesized that CAV1 overexpression may be associated with decreased expression of miR-124 in renal cell carcinoma. As for the effector of CAV1 downstream, CAV1 can activate AKT through Interactions with and Inhibition of PP1 and PP2A in prostate cancer cells [34]. In addition, previous report indicates that PP2A is down-regulated in kidney cancer [35]. These reports suggest that PP2A may be a potential downstream target of CAV1. CAV1 can also interact with AR to promote AR transcriptional activity [36]. We will further explore the potential mechanisms of CAV1 overexpression and its effectors in kidney cancer in future studies. In our study, we found that CAV1 expression was elevated in kidney cancer cells and tissues, consistent with many previous studies in other tumor types. Moreover, we discovered that high CAV1 expression was associated with poor DFS and could serve as diagnostic indicator in ccRCC patients with different clinicopathological stages. We hypothesized that elevated CAV1 may play an important role in the development and progression of renal cell carcinoma. In order to verify our speculation, we had done a variety of functional experiments in renal carcinoma cell lines. The results of functional experiments show that CAV1 promotes the migration and invasion of renal cell carcinoma, indicating that CAV1 may affect the biological behavior of renal cell carcinoma. Due to the existence of intrinsic or acquired drug resistance mechanisms, resistance to first-line drug sunitinib still remains a major problem to be solved in clinical therapy, and CAV1 is reported to be involved in drug resistance in a variety of tumors. Unfortunately, there is no study on the role of CAV1 in sunitinib resistance mechanisms in renal cancer. We established sunitinib-resistant cell lines in kidney cancer cells with continuous low-dose stimulation in conjunction with intermittent high-dose pulses of sunitinib. The expression of CAV1 in sunitinib-resistant cell lines was significantly higher than that in parental cell lines. Plasmid-mediated overexpression of CAV1 significantly increased resistance to sunitinib in parental and drug resistant cell lines, while knockdown of CAV1 expression did not increase the sensitivity of cells to sunitinib. Due to inconsistent with our expected
3.7. The effects of CAV1 knockdown or overexpression on sunitinib sensitivity and cells viability To investigate the role of CAV1 in sunitinib resistance procedures, we successfully overexpressed or knocked down CAV1 in sunitinibresistant cell lines and parental cell lines. We then examined the cells viability in sunitinib-resistant and parental cell lines at various sunitinib concentrations after CAV1 being overexpressed or knocked down, and the IC50 values of different intervention groups cells were listed in Table 2. As shown in Fig. 7A+C, the cell lines of CAV1 overexpression possessed a higher cell viability compared with the corresponding control cell lines. Contrary to our desired outcome, CAV1 knockdown did not enhance sunitinib sensitivity in ccRCC cells (Fig. 7B, D). At some different sunitinib concentrations, CAV1 knockdown cells even revealed a clearly more sunitinib-resistant phenotype rather than the desired sunitinib-sensitive phenotype. We speculated that the reduced cells proliferation rate of CAV1 knockdown cells resulted in insensitivity of these cells to sunitinib and other drugresistant genes might continue to maintain cell-resistant phenotype in the case of CAV1 knockdown. These results suggested that CAV1 overexpression might contribute to cellular tolerability of sunitinib. 3.8. CAV1 promotes the migration and invasion of ccRCC cells in vitro To determine how CAV1 contributes to aggressiveness of ccRCC, we first examined whether CAV1 increased cell migration and invasion in vitro, the critical features of tumor metastatic phenotype. Given the high CAV1 expression in all ccRCC cell lines detected, we first selected a CAV1 si-RNA knockdown method. Two CAV1 si-RNA oligonucleotide sequences were used, resulting in consistent CAV1 knockdown (Fig. 8A). Scratches of 786-O and ACHN cells healed more rapidly than corresponding CAV1 knockdown cells in wound-healing assays (Fig. 8C). 786-O and ACHN cells also possessed a faster invasion capacity than corresponding CAV1 knockdown cells in transwell assays (Fig. 8E). Next, we transferred the CAV1 plasmid to 786-O and ACHN cells, resulting in overexpression of CAV1 (Fig. 8B). Scratch assays and transwell invasion experiments demonstrated that cells migration and invasion of CAV1-expressing cells were faster compared with the corresponding control cell lines (Figs. 8D and F). These data demonstrated that CAV1 could promote the migration and invasion capacity of ccRCC cells. 4. Discussion Existing understanding about the molecular mechanisms underlying disease progression and sunitinib resistance in ccRCC is still incomplete. Cancer metastasis remains the major cause of death in ccRCC patients. Receptor tyrosine kinase inhibitors (RTKI), including sunitinib, are currently the first-line therapeutic agents for metastatic and advanced renal cell carcinoma. However, patients who are completely or continuously sensitive to RTKI are very rare, and even those who are initially sensitive to RTKI will subsequently develop and progress into RTKI resistance after an average of 6–15 months of treatment [14]. Thus, cancer metastasis and drug resistance remain the major clinical problems in ccRCC. However, the specific molecular mechanisms of cancer metastasis and drug resistance are still not very clear in ccRCC. Cumulative evidence suggests that CAV1 is significantly overexpressed in a variety of human malignant cancers, including cancers of liver [4], bladder [9], prostate [7], brain [15], suggesting that it may play an important role in the initiation and progression of these tumors. Previous study has confirmed that overexpression of CAV1 is associated with the recurrence of progressive prostate cancer [16]. CAV1 upregulation is associated with poor prognosis and enhanced metastatic capacity in lung cancer [17]. CAV1 contributes to tumor 9
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results, we hypothesized that other drug-resistant genes might continue to maintain cell-resistant phenotype in the case of CAV1 knockdown. However, there is a drawback in our study that the role of CAV1 in the animal model of kidney cancer and the mechanism of sunitinib resistance in vivo remain unclear. In future studies, we will explore the biological effects of CAV1 and the mechanism of sunitinib resistance in vivo, aiming to elucidate the role of CAV1 in the progression of renal cell carcinoma and sunitinib resistance. In conclusion, our study confirms that CAV1 is elevated in RCC tissues and cell lines as well as in sunitinib-resistant cell lines and high CAV1 expression can serve as a useful molecular marker for the diagnosis and prognosis of ccRCC patients. Moreover, our findings confirm that CAV1 promotes the migration and invasion of renal carcinoma cells and increases the tolerance of renal carcinoma cells to sunitinib. Although the in vivo function of CAV1 needs to be evaluated, the data derived from our experiments strongly support the idea that the development of CAV1-targeting drug may increase the efficiency of ccRCC treatment and improve the therapy outcome for ccRCC patients, especially those presenting with advanced and metastatic ccRCC. Statement of conflict of interest The authors state that they have no conflicts of interest. Acknowledgements This research was supported by the National Natural Science Foundation of China (Grant No. 81672528, 81672524 & 81272560), the Program for New Century Excellent Talents in University from the Department of Education of China (NCET-08-0223), the National High Technology Research and Development Program of China (863 Program) (2012AA021101). Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at doi:10.1016/j.yexcr.2017.07.004. References [1] R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics, 2017, CA: Cancer J. Clin. 67 (1) (2017) 7–30 (PubMed PMID: 28055103). [2] S.F. Tavazoie, C. Alarcon, T. Oskarsson, D. Padua, Q. Wang, P.D. Bos, et al., Endogenous human microRNAs that suppress breast cancer metastasis, Nature 451 (7175) (2008) 147–152 (PubMed PMID: 18185580. Pubmed Central PMCID: 2782491). [3] I. Tamaskar, T.K. Choueiri, L. Sercia, B. Rini, R. Bukowski, M. Zhou, Differential expression of caveolin-1 in renal neoplasms, Cancer 110 (4) (2007) 776–782 (PubMed PMID: 17594718). [4] E.Y. Tse, F.C. Ko, E.K. Tung, L.K. Chan, T.K. Lee, E.S. Ngan, et al., Caveolin-1 overexpression is associated with hepatocellular carcinoma tumourigenesis and metastasis, J. Pathol. 226 (4) (2012) 645–653 (PubMed PMID: 22072235). [5] G. Yuan, I. Regel, F. Lian, T. Friedrich, I. Hitkova, R.D. Hofheinz, et al., WNT6 is a novel target gene of caveolin-1 promoting chemoresistance to epirubicin in human gastric cancer cells, Oncogene 32 (3) (2013) 375–387 (PubMed PMID: 22370641). [6] O.M. Tirado, C.M. MacCarthy, N. Fatima, J. Villar, S. Mateo-Lozano, V. Notario, Caveolin-1 promotes resistance to chemotherapy-induced apoptosis in Ewing's sarcoma cells by modulating PKCalpha phosphorylation, Int. J. Cancer 126 (2) (2010) 426–436 (PubMed PMID: 19609943. Pubmed Central PMCID: 2794946). [7] G. Ayala, M. Morello, A. Frolov, S. You, R. Li, F. Rosati, et al., Loss of caveolin-1 in prostate cancer stroma correlates with reduced relapse-free survival and is functionally relevant to tumour progression, J. Pathol. 231 (1) (2013) 77–87 (PubMed PMID: 23729330. Pubmed Central PMCID: 3978784). [8] C.C. Ho, S.H. Kuo, P.H. Huang, H.Y. Huang, C.H. Yang, P.C. Yang, Caveolin-1 expression is significantly associated with drug resistance and poor prognosis in advanced non-small cell lung cancer patients treated with gemcitabine-based chemotherapy, Lung Cancer 59 (1) (2008) 105–110 (PubMed PMID: 17850918). [9] T. Qayyum, G. Fyffe, M. Duncan, P.A. McArdle, M. Hilmy, C. Orange, et al., The interrelationships between Src, Cav-1 and RhoGD12 in transitional cell carcinoma of the bladder, Br. J. Cancer 106 (6) (2012) 1187–1195 (PubMed PMID: 22353809. Pubmed Central PMCID: 3304420). [10] L. Liu, H.X. Xu, W.Q. Wang, C.T. Wu, T. Chen, Y. Qin, et al., Cavin-1 is essential for the tumor-promoting effect of caveolin-1 and enhances its prognostic potency in pancreatic cancer, Oncogene 33 (21) (2014) 2728–2736 (PubMed PMID: 23770857).
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