PTPN3 Inhibits the Growth and Metastasis of Clear Cell Renal Cell Carcinoma via Inhibition of PI3K/AKT Signaling

The underlying molecular mechanism driving clear cell renal cell carcinoma (ccRCC) progression is still to be explored. The significant downregulation of protein tyrosine phosphatase nonreceptor type 3 (PTPN3) expression in the tumor tissues suggested its protective role in ccRCC progression. IHC analysis of PTPN3 protein in 172 ccRCC tissue revealed that PTPN3 was an independently favorable prognostic factor for progression-free survival (P = 0.0166) and overall survival (P = 0.0343) of patients. The ccRCC cell lines SN12C, 1932, ACHN, and Caki-1 were used to evaluate, both in vitro and in vivo, the biological roles of PTPN3. We observed that overexpression of PTPN3 significantly inhibited the proliferation, migration, and invasion of ccRCC cells. In contrast, the knocking down of PTPN3 elicited opposite effects. Overexpressing PTPN3 inhibited xenograft tumor growth and lung metastasis displayed by the in vivo mice models. PTPN3 inhibited tumor cell motility by suppressing the phosphorylation of AKT, and subsequently inactivating the PI3K/AKT signaling pathway of renal cell carcinoma cells. Furthermore, the inhibition of phospho-AKTThr308 and phospho-AKTSer473 reversed PTPN3-induced silencing in tumor cell migration. Our work revealed that the overexpression of PTPN3 could suppress kidney cancer progression by negatively regulating the AKT signaling pathway, and served as a favorable prognostic factor in patients with ccRCC. Our findings provided insight that PTPN3 could be a potential target for therapy aiming to inhibit the malignant behaviors of ccRCC. Implications: PTPN3 is an independent favorable prognostic factor for patients with ccRCC and could be a potential target for therapy aiming to inhibit the malignant behaviors of ccRCC.


Introduction
Kidney cancer represents about 4% of all new cancer diagnoses (1). The most common form of kidney cancer arises from renal epithelium, termed as renal cell carcinoma (RCC; ref. 2). As the most common histologic subtype, clear cell renal cell carcinoma (ccRCC) accounts for 70%-75% of all RCC cases (3). The worldwide incidence and mortality rate of ccRCC has been estimated rising by 2%-3% per decade (4). In spite of the fact that the small renal masses have been more frequently detected in recent year, one third of patients with ccRCC will suffer from metastatic diseases after the diagnosis (5). The treating of metastatic ccRCC still remains a challenge as the underlying mechanism(s) is yet to be elucidated (6,7).
The PI3K/protein kinase B (also named AKT) signaling pathway has been shown to regulate a series of cellular functions in various types of cancer, including proliferation, autophagy, apoptosis, cell cycle, and metastasis (8,9). Consequently, several potential therapeutic targets of this signaling pathway have been proposed (10), which have also been found to be activated and playing important roles in ccRCC progression (11,12).
Protein tyrosine kinases and protein tyrosine phosphatases (PTP) reversibly regulate the phosphorylation of tyrosine proteins, acting as a molecular switch for the regulation of various biological processes (13). Aa a membrane-associated nonreceptor of PTP, PTPN3 contains a C-terminal phosphatase domain, a middle PDZ domain and an N-terminal FERM domain (14). These three domains involve in the regulation of cellular proliferation and growth (15). As a tyrosine phosphatase, PTPN3 could dephosphorylate several substrates, for example, the EGFR pathway substrate 15 (13). However, its role as a nonreceptor protein tyrosine kinase is still unclear.
Accumulating evidence has suggested an equivocal role of PTPN3 in the progression of a variety of human cancers (13,14,16). Li and colleagues found that the ectopic expression of PTPN3 in non-small cell lung cancer cells suppressed cellular motility, proliferation, and tumor growth via the regulation of EGFR endocytic trafficking, degradation, and signaling (13). On the contrary, Hou and colleagues showed that PTPN3 may increase malignant growth by cooperating with its substrate, p38g (16). Gao and colleagues also found that numerous activating mutations and high expression levels of PTPN3 were associated with intrahepatic cholangiocarcinoma recurrence (14). Thereby, the exact role of PTPN3 in ccRCC remains undetermined.
In this study, through the findings of various investigations, we reported that PTPN3 could suppress ccRCC progression by negatively regulating the AKT signaling pathway, providing insight that PTPN3 could be a potential target for therapy aiming to inhibit the malignant behaviors of ccRCC.

Cell lines and culture
Originally bought from ATCC, ccRCC cell lines SN12C, 1932, Caki-1, ACHN were routinely maintained in liquid nitrogen in our laboratory (17,18). The cell lines were cultured in DMEM or 1640 (Gibco) supplemented with 10% FBS (Gibco) with 100 IU/mL of streptomycin and 100 mg/mL of penicillin in a humidified atmosphere of 5% CO 2 at 37 C as described previously (19). We used the short tandem repeat (STR) testing to authenticate all cells and make sure that they were maintained Mycoplasma-free.

Patient tissue samples
Twelve ccRCC tissues and matched paracancerous tissues were retrieved from the Department of Urology, Sun Yat-sen University Cancer Center (SYSUCC, Guangzhou, China) and stored at À 80 C before use for quantitative real-time reverse-transcription PCR (qRT-PCR) analysis. Another cohort of 172 formalin-fixed, paraffinembedded primary ccRCC specimens collected from the Department of Pathology, SYSUCC was used for IHC evaluation. Written informed consent was obtained from all patients and the study was approved by the ethics committees of SYSUCC as described previously (19).

RNA isolation and qRT-PCR
We used TRIzol reagent (Invitrogen) to isolate total RNA. Complementary DNA (cDNA) was synthesized using a reverse transcription kit (K1622, Thermo Fisher Scientific) following the manufacturer's instructions as described previously (19). qRT-PCR analysis was performed using the SYBR Green PCR Kit (Novazym), and b-actin PTPN3 mRNA and protein expression levels in ccRCC tissues and cell lines. A, qRT-PCR analysis of PTPN3 mRNA expression levels in ccRCC matched tissues (n ¼ 12). The mRNA levels of PTPN3 in ccRCC tissues were lower than the paired normal tissues (P ¼ 0.0006, paired t test). B, Levels of PTPN3 protein expression in ccRCC tissue chip are shown under both low and high magnifications of a light microscope; low PTPN3 level correlated with shorter overall survival and progressionfree survival in patients with ccRCC. C, In ccRCC tissue chip from our hospital, the overall survival (OS) and progression-free survival (PFS) rate were significantly higher in the higher PTPN3 group rate. D, Similarly, using data from the TCGA database, we observed that the overall survival and progressionfree survival rate was significantly higher in the higher PTPN3 group.

MTS assay
The cells were plated into 96-well plates at a density of 2,000 cells/ well in 200 mL normal culture medium. Cell growth was determined using MTS (Cell Titer 96 Aqueous One Solution Cell Proliferation Assay solution; Promega), as described previously (19). Every day at the same time, 10 mL of the MTS reagents were added to 100 mL culture medium per well and incubated for 2-4 hours at 37 C. The microplate reader (Bio-Tek EPOCH2) was used to read the OD490 of these cells seeded into 96-well plates. All experiments were repeated three times independently.

Clonogenic assay
Equal numbers of cells were seeded at a density of 500 cells/mL into 6-well plates and incubated for 10 days at 37 C. At the end of the experiments, the cells were stained with 0.1% crystal violet for 15 minutes. We used the microscope (NIKON ECLIPSE 80i) to count the number of positive colonies, those with >50 cells.

Lentiviral transduction studies
PTPN3-overexpressing plasmid or vector were purchased from Genecopoeia Co. Ltd. as described previously (19). Lentiviruses were produced in 293 T cells cotransfected with lentiplasmid and packing plasmids using the X-treme GENE DNA transfection reagents (Roche). The cells were first incubated with infectious lentiviruses for 12 hours and then exchanged to incubation with fresh medium for another 12 hours, after which they were selected with puromycin (2 ng/L) for 3 days and validated by qRT-PCR and immunoblotting (19).

Migration and invasion assays
A total of 5 Â 10 4 cells of ACHN, 3 Â 10 4 cells of Caki-1, 4 Â 10 4 cells of SN12C, or 2 Â 10 4 cells of 1932 were seeded in a serum-free medium in the top chambers (Corning) with 700 mL of DMEM with 10% FBS in the bottom chambers. After culturing for 24 hours, the cells were fixed with methanol and then they were stained with 0.1% crystal violet for 30 minutes at room temperature. For invasion assays, the top chamber membranes were coated with Matrigel (Corning, Life Sciences). Three random fields per well were observed, and cells were counted under a microscope (NIKON ECLIPSE 80i) as described previously (19). The inhibitor of AKT-MK-2206 (1 mmol/L) was purchased from Selleck Chemicals. Both experiments were performed in triplicates.

Wound healing assays
Cells were seeded into 6-well plates (Corning) and cultured to a 90% confluent followed by 24-hour starvation in a serum-free medium. We used the sterile 200-mL tip to create artificial wounds in the cell monolayer and the floating cells were removed by washing with PBS as described previously (19). We used an inverted microscope (OLYM-PUS IX73) to capture respective images at 0, 6, 12, 24, 48, 72, and 96 hours.

IHC staining
Rabbit anti-PTPN3 antibody (1:100 dilution, #GTX54572, Gen-eTex) was used, and the IHC staining results were assigned a mean score considering both the intensity of staining and the proportion of tumor cells with an unequivocal positive reaction as described previously (19). Two pathologists without prior knowledge of patient data assessed each section independently. The percentage of stained cells was scored as follows: 0% as 0; 1%-10% as 1; 11%- 50% as 2; 51%-80% as 3; 81%-100% as 4, and the intensity of staining: no staining as 0; weak staining as 1; moderate staining as 2; strong staining as 3. Then multiplying the two was the score of each tissue (score range: 0-12).

Animal experiments
Female athymic mice between 4 and 6 weeks of age were obtained from Beijing Vital River Laboratory Animal Center. Mice were cared in accordance with the principles and procedures outlined in the Institutional Animal Care and Use Committee of SYSUCC as described previously (19). A total of 8 Â 10 6 /0.15 mL SN12C cells stably expressing scrambled shRNA control or shPTPN3 were injected subcutaneously into the mice. Ten days after the injection, the mice were sacrificed and the tumors were excised and measured. We injected SN12C kidney tumor cells persistently expressing scrambled shRNA control or shPTPN3 (1.5 Â 106 cells/0.1 mL PBS) into the lateral tail vein of nude mice to get lung metastases. After eight weeks, the lungs were resected, weighed, and photographed. We counted the number of metastatic nodules on the surface of the lungs visually after staining with picric acid. Paraformaldehyde was used to fix the lungs for paraffin sections and then hematoxylin and eosin were used to stain the lungs with a standard histologic protocol.

Statistical analysis
The SPSS V.14 software package was used to perform statistical data analyses. P < 0.05 was considered significant. Data are presented as the mean AE SD. Multivariate analyses were performed using the Cox proportional hazards model. Survival curves were constructed using the Kaplan-Meier method and compared using the log-rank test as described previously (19). We used t test to compare PTPN3 mRNA levels between normal and tumor samples from the kidney renal clear cell carcinoma dataset of The Cancer Genome Atlas (TCGA). We have uploaded the key raw data onto the Research Data Deposit (RDD) platform (www.researchdata.org. cn) to validate the authenticity of this article, with the approval number as RDDB2019000767.

Reduced expression of PTPN3 correlate with poor prognosis in patients with ccRCC
We investigated the expression of PTPN3 in ccRCC tissues. Quantitative real-time PCR analysis showed that PTPN3 mRNA was dramatically lower in tumoral tissues as compared with their corresponding adjacent normal renal tissues in the 12 matched human ccRCC tissues (Fig. 1A). We also performed IHC staining in 172 human ccRCC samples (Fig. 1B) to evaluate the expression level of PTPN3 in ccRCC tissue. PTPN3 mainly expressed in the cytoplasm of tumor cells in those IHC-stained human ccRCC tissue samples. The association between PTPN3 expression and clinicopathologic features to survival results are summarized in Tables 1  and 2. Patients with tumors having high PTPN3 expression (cut-off score > 6) had significantly longer progression-free survival (PFS) and overall survival (OS) than those with low PTPN3 expression tumors ( Fig. 1C; P ¼ 0.0343 vs. P ¼ 0.0166). Multivariate analyses revealed that high PTPN3 expression was an independent and favorable prognostic indicator for PFS (P ¼ 0.047) in all patients ( Table 2).
We also analyzed the gene expression data of patients with ccRCC in the TCGA database. PTPN3 mRNA was found to be significantly lower in all tumor samples than in paired normal tissues ( Supplementary  Fig. S1A-S1C; P < 0.05). For separating the cases into low and high PTPN3 expression groups, the mean expression level was used as the cut-off value. Kaplan-Meier survival analyses of TCGA data also showed that lower expression of PTPN3 was associated with poorer survival in ccRCC (Fig 1D; P < 0.0001). These observations suggest that low levels of PTPN3 may act as a novel prognostic factor for patients with ccRCC.

Knocking down PTPN3 promotes ccRCC cell growth in vitro and in vivo
As is known to us, 1932 and SN12C were derived from primary human RCC, Caki-1 was a widespread model line of metastatic ccRCC, and ACHN was model line of metastatic papillary RCC (20). We transfected SN12C and 1932 cells with siRNA for PTPN3 (si1# and si2#) or negative control siRNA ( Fig. 2A and B). PTPN3 suppression significantly increased RCC cell colony formation and proliferation (Fig. 2C) abilities of these two cell lines (Supplementary Fig. S2). Furthermore, PTPN3 was stably overexpressed in SN12C, Caki-1, and ACHN cell lines (Fig. 2D) and overexpressing PTPN3 effectively suppressed cell proliferation (Fig. 2E) and decreased colony formation ability (Fig. 2F). In addition, the overexpression of PTPN3 significantly inhibited tumor growth in nude mice (Fig. 2G and F). Taken together, these results suggested that PTPN3 can act as a tumor suppressor in the development of RCC.

PTPN3 inhibits ccRCC cell migration, invasion, and metastasis
We performed wound-healing, transwell migration, and invasion assays to examine whether PTPN3 influences renal cell carcinoma cellular mobility. Findings from these assays revealed that the knockdown of PTPN3 with siPTPN3 increased the migration and invasion of renal cell carcinoma cells transfected (Fig. 3A, B, and E). Accordingly, the overexpression of PTPN3 significantly decreased the migration and invasion ACHN and SN12C cells (Fig. 3C, D, and F). Furthermore, the tail veins and lung metastatic in vivo experiments in nude mice showed that the number of lung metastatic tumors were significantly fewer in the SN12C-PTPN3 group than the control group (P < 0.05; Fig. 3G and H). Taken together, these results showed an important inhibitory role of PTPN3 in the migration, invasion, and metastasis of ccRCC cells.

PTPN3 inhibits ccRCC cellular motility through suppression of PI3K/AKT signaling
Protein kinase B (also named AKT) is an important mediator of cell proliferation, apoptosis, autophagy, and metastasis. Here, we found that silencing PTPN3 in the 1932 and SN12C cells could enhance the expression of phospho-AKT Thr308 and phospho-AKT Ser473 (Fig. 4A). Conversely, the expression of phospho-AKT Thr308 and phospho-AKT-Ser473 in 1932 and SN12C cells were decreased by overexpressing PTPN3 (Fig. 4A). Furthermore, we found that treatment with MK-2206 decreased phospho-AKT Thr308 and phospho-AKT Ser473 in SN12C-siPTPN3 and 1932-1-siPTPN3 cells (Fig. 4B). These results suggest that PTPN3 had some negative effects on the PI3K/AKT signaling pathway in RCC cells. PTPN3 seemed increased by the treatment of AKT inhibition for 1932 cell line, while this experimental  phenomenon could not be repeated for SN12C cell line. In addition, we found that AKT inhibition by MK-2206 impaired the migration and invasion of SN12C-siPTPN3 and 1932-siPTPN3 cells (Fig. 5A and B), as well as the proliferation of SN12C-siPTPN3 and 1932-siAKT cells ( Fig. 5C and D). These results illustrate that the knockdown of PTPN3 stimulated RCC cells' growth, migration, and invasion through the activation of the AKT pathway.

Discussion
Although metastases cause the most death in patients with ccRCC, the detailed mechanisms of metastasis remain unclear (6,7). In this study, we revealed that the expression of PTPN3 was downregulated in ccRCC cell lines and tissue samples. Also, the overexpression of PTPN3 inhibited renal cell carcinoma cell growth, migration, and invasion, and resulted smaller subcutaneous tumors and fewer pulmonary metastatic tumors in nude mice. Furthermore, lower expression of PTPN3 protein in ccRCC tissues could independently predict poor overall survival and progression-free survival in ccRCC. From this preliminary study, we found that PTPN3 could be a powerful conservator for antimetastasis in ccRCC.
The PI3K/AKT pathway has been shown to play important roles in a variety of biological processes, and the dysfunction of the AKT signaling was associated with diseases such as cancer, diabetes, and autoimmune (21,22). PIP3 recruits AKT and PDK1 and other pleckstrin homology domain-containing proteins to the membrane of cells. Phosphorylating at Thr308 by PDK1 and at Ser473 by the mTOR complex 2 (mTORC2) increases the kinase activity of AKT (23). Directly and indirectly, AKT phosphorylates many downstream proteins to enhance cell survival and growth including the GSKs, p27KIP1, FoxO transcription factors, MDM2, and BAD (24). In this study, we discovered that PTPN3 attenuated AKT activity in RCC by downregulating both phospho-AKT Thr308 and phospho-AKT Ser473 .
Because AKT was found to be a crucial regulator of cell proliferation, growth, and survival in different kinds of tumors (25), our findings may be applicable to not only RCC, but also other cancers that are driven by the increase of AKT signaling. Targeting AKT pathways through PTPN3 may be a major goal to develop therapeutics. Although our results suggested that PTPN3 could inhibit tumor progression in RCC, in other cancers, different mechanisms could be involved (26)(27)(28). What was more, Li and colleagues found that tyrosine-phosphorylated Src and DAAM1 could be dephosphorylated by the catalyzing of PTPN3 and thus suppresses lung cancer cell invasiveness (29). However, Yuan and colleagues found that PTPN3 profoundly potentiates TGFb signaling independent of its phosphatase activity and thus acts as a tumor suppressor in liver cancer (30). In our study, we found that PTPN3 might inhibit the progression of RCC via the PI3K/AKT signaling pathway because PTPN3 could decrease the expression of both phospho-AKT Thr308 and phospho-AKT Ser473 in RCC cells. However, whether PTPN3 was able to dephosphorylate and directly act on AKT was still to be validated in further researches.
The mechanism between AKT and cancer has been reported in many other studies. For instance, some researchers found that m6A methylation could affect AKT activity in adult T-cell leukemia (AML; ref. 31) and endometrial cancer (26); CITED2 could activate the nucleolin-AKT pathway and thus promoted prostate cancer metastasis (32); SMAD and AKT pathways interacted to confer prooncogenic responses to TGFb and drove breast cancer metastasis (33). Some studies further reported the potential mechanisms between AKT pathways and renal cell cancer. AKT was found to positively regulate rasfonin-enhanced autophagy and caspase-dependent apoptosis pri-marily through affecting the glycolytic pathway in renal cancer cells (34). However, the upstream potential mechanism of AKT was rarely reported. Some researchers found that downregulating PIK3R1 in RCC promoted the migration, propagation, stem-like phenotype, and EMT in RCC cells via the AKT/GSK3b/CTNNB1 pathway and may lead to the progression of RCC (35). In this study, we found that PTPN3 was actually an inhibitor for AKT pathway in ccRCC upon controlling cellular proliferation and motility.

Conclusions
PTPN3 inhibits RCC cells' growth, migration, and invasion via downregulation of the AKT pathway.

Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.