Expression and Functional Identication of INPP4B in Gallbladder Cancer Patients and Gallbladder Cancer Cells

Purpose: Inositol polyphosphate 4-phosphatase type II (INPP4B) is a negative regulator of PI3K-Akt signaling pathway and plays a contradictory role in different types of cancers. However, its biological role in human gallbladder cancer (GBC) remain unclear. Here we aimed to investigate the expression, clinical signicance and biological function of INPP4B in GBC clinical dates and GBC cell lines. Methods: The INPP4B protein expression levels in gallbladder cancer tissues and normal gallbladder tissues were detected by immunohistochemistry, and the clinical signicance of INPP4B was analyzed. Knockdown and overexpression of INPP4B on GBC-SD and SGC-996 cells were used to identify INPP4B function in vitro, using cell proliferation assay, clonogenic assay, apoptosis detection, cratch wound-healing assay and transwell assay. Results: INPP4B was up-regulated in human GBC tissues compared with normal gallbladder tissues, and was related to histopathological differentiation. Here, we observed that INPP4B was highly expressed in high-moderate differentiated compared to low-undifferentiated. Additionally, we found that INPP4B expression was not associated with overall survival in GBC patients and was not an independent prognostic factor. Furthermore, when we stratied the relationship between INPP4B expression and prognosis of GBC from histopathological differentiation, we found that INPP4B played a contradictory role in GBC progression at different degrees of differentiation. In addition, INPP4B knockdown inhibited tumorigenicity in vitro, and INPP4B overexpression induced tumorigenicity in vitro, which may play a role as an oncoprotein. Conclusions: These ndings implicated that INPP4B may play a dual role in the prognosis of GBC with different degrees of differentiation, and might act as an oncogene in gallbladder cancer cells.


Introduction
Gallbladder cancer (GBC) is the most common malignant tumor of the biliary system, which has the characteristics of gender, geographical location and race difference [1,2]. In recent years, although the diagnosis and treatment of GBC has been greatly improved, the prognosis of GBC patients has not been signi cantly improved, with a ve-year survival rate of less than 5% and an average survival of 6 months [3]. Therefore, elucidating the molecular mechanisms of GBC progression is critical to facilitate the development of new diagnostic and therapeutic strategies.
PI3K-Akt signaling pathway plays an important role in tumor cell proliferation, differentiation, angiogenesis, invasion and metastasis [4,5]. Hyper-activation of PI3K-Akt signaling has been shown to be the driving factor of tumor initiation and progression [6,7]. PI3K-Akt signaling pathway has been considered as a therapeutic target for a variety of malignant tumors, and many clinical trials have been conducted to investigate the therapeutic effects of PI3K-Akt pathway inhibitors on human cancer [8].
Recently, Inositol polyphosphate-4-phosphatase type II (INPP4B), as a phosphoinositide phosphatase, has been proved to be a negative regulator of the PI3K-Akt signaling and plays a role of tumor suppressor gene (TSG) [9][10][11][12]. Low expression of INPP4B in several type of cancers is associated with poor clinical outcomes [13][14][15]. Knockdown of INPP4B could enhance breast cancer, melanoma and prostate cancer cell lines proliferation and migration, suggesting its TSG role in these cancer cells [11,15,16]. However, recently, some studies have reported that INPP4B is highly expressed in colon cancer, acute myeloid leukemia and melanoma, which plays a role of oncogene and has a positive correlation with poor clinical outcomes [17][18][19]. Even within the same tumor, different researchers have come to different conclusions about the role of INPP4B in oncogene and TSG [16,[18][19][20]. Above the previous studious indicate that the expression and functional role of INPP4B remains controversial in cancers, which seems to be related to tumor types. However, to date, the expression level, clinical prognostic value and biological functional of INPP4B in GBC have not been studied. To help comprehensive understand the potential value of INPP4B in GBC, it will be very useful to detect its expression level, evaluate its clinical prognostic signi cance and study its cell function in vitro.
In the present study, we detected the expression of INPP4B in GBC tissues and non-tumorous tissues by immunohistochemistry. We found that the expression of INPP4B in GBC tissues was signi cantly higher than that in non-tumorous tissues. The INPP4B protein expression was decreased in low-undifferentiated of GBC tissues but increased in high-moderate differentiated tissue. In addition, high INPP4B expression was associated with a favourable prognosis in patients high-moderate differentiated, while poor prognosis in patients with low-undifferentiated. We further explored the biological function of INPP4B in GBC cells in vitro. We found that INPP4B knockdown inhibited tumorigenicity, while INPP4B overexpression induced tumorigenicity in vitro. These results suggested that INPP4B plays a critical role in GBC and may provide a potential target for the treatment of GBC.

Patients and tissue specimens
In the present study, a total of 127 GBC patient tissues and 47 non-tumorous tissues were collected from the Armed Police Corps Hospital of Anhui (Hefei, China) using for analysising INPP4B expression by immunohistochemistry staining. Detailed clinicopathological parameters were described in Table 1.
Patients did not receive any anticancer treatment prior to surgery.

Immunohistochemistry (IHC)
Immunohistochemistry (IHC) was used to detect the expression of INPP4B in 4-µm formalin-xed para n-embedded human GBC tissues and non-tumorous tissues. The IHC process and staining evaluation were performed as previously reported [21,22]. The tissue sections were incubated with INPP4B primary antibody (Abcam, ab81269, 1:50) and negative controls with normal rabbit IgG (Beyotime Institute of Biotechnology, A7016). The immunohistochemical staining results were evaluated by two independent pathologists who did not know the patient's clinical information. The immunoreactivity score (IRS) were determined by "staining intensity × staining percentage". The specimen Cell culture and lentivirus infection GBC-SD and SGC996 GBC cell lines were obtained from Genechem (Shanghai, China) and cultured in RPMI1640 medium supplemented with 10% FBS, penicillin (100 U/ml) and streptomycin (100 µg/ml) in a 5% CO2 humidi ed incubator at 37 °C. In order to establish GBC-SD cell and SGC996 cell with stable  overexpression of INPP4B, the two cell lines were infected with INPP4B overexpression lentiviral (GV492-INPP4B, OEINPP4B) and control lentiviral (GV492, OECtrl, purchased from Genechem, Shanghai, China) respectively. In addition, for establishing stable INPP4B knockdown GBC-SD cell and SGC996 cell, the two cell lines were infected with shRNA INPP4B lentiviral (GV248-shINPP4B) and control lentiviral (GV248-shCtrl, purchased from Genechem, Shanghai, China). The target sequence of shINPP4B was as 5'-CCATCTGAGTATCCCATCTAT-3′. Overexpression and knockdown e ciency of target genes were detected by q-PCR.
Cell proliferation assay and clonogenic assay Cell proliferation assay and clonogenic assay were employed to measure the role of INPP4B on GBC cell viability and proliferation capacity. The detailed cell proliferation assay processes was performed as described previously [23,24]. Brie y, different lentivirus (OEINPP4B, OECtrl; shINPP4B, shCtrl) infection GBC cell lines (GBC-SD and SGC-996) were seeded into 96-well plates (about 2000 cells/well) in sextuple, and cell viability was determined by MTT (Genview, JT343) at each 24 h interval following the manufacturer's instructions. The number of viable cells was determined by measuring absorbance at OD490 nm using a Universal Microplate Reader (BioTek Instruments, Inc.). Each experiment was performed in three times. The detailed colony formation assay processes was performed as described previously [25,26]. In short, different lentivirus infection GBC cell lines were seeded into 6-well plates (about 800 cells/well) and the medium was replaced every three days. After two weeks, the colonies were xed by 4% polyoxymethylene, stained with Giemsa, and counted by inverted microscope. Each assay was repeated in three times.

Apoptosis assay
The detailed process of apoptosis analysis has been described in previous studies [27]. In brief, according to the manufacturer's instructions, the apoptosis of different lentivirus infection GBC cell lines was analyzed using the Annexin V-APC apoptosis detection kit (eBioscience, 88-8007). The apoptosis rate was analyzed by ow cytometry (FACSCalibur; BD Biosciences).
Scratch wound-healing assay and transwell assay Cell migration and invasion ability were assessed by scratch wound-healing assay and transwell assay and performed as previously described [28,29]

Results
INPP4B is increased in GBC tissues, and serves as a prognostic factor INPP4B protein expression in tumors are still controversial and has not been studied in GBC tissues. We analyzed the protein expression of INPP4B in 127 GBC tissue samples and 47 non-tumorous tissues by immunohistochemistry. As a result, INPP4B staining was mainly located in the cell cytoplasm, and the positive rate was signi cantly higher in GBC tissues than in non-tumorous tissues (p = 0.008, data not shown). Representative images are shown in Fig. 1A. The results revealed that INPP4B was highly expressed in GBC and was mainly located in the cytoplasm.
In addition, when we further elucidated the correlation between INPP4B expression and GBC clinicopathological parameters, we found that INPP4B expression was closely associated with histopathological differentiation, and the immunoreactivity score (IRS) of high-moderate differentiated tissues was markedly higher than that of low-undifferentiated tissues (Table 1 and Fig. 1B). These data suggested that INPP4B may play an important role in the progression of GBC.
Based on the above ndings, we speculated that INPP4B might be related to GBC prognosis. However, Kaplan-Meier analysis and log-rank test revealed that INPP4B expression was not associated with overall survival (OS) in GBC patients. Multivariate analysis showed that INPP4B was not an independent predictor factor ( Fig. 1C and Table 2). However, when we strati ed the relationship between INPP4B expression and prognosis of GBC patients in terms of histopathological differentiation, we got some interesting ndings. In high-moderate differentiation group, we found that GBC patients with INPP4B + showed better prognosis than that of patients with INPP4B − (Fig. 1D); while in low-undifferentiated group, we found that GBC patients with INPP4B + showed worse prognosis than that of patients with INPP4B − (Fig. 1E). These results indicate that INPP4B plays a contradictory predictor role in GBC progression with different histopathological differentiation.

INPP4B regulates GBC cells proliferation in vitro
Given the high expression of INPP4B in GBC tissue and its correlation with the clinical prognosis of GBC patients, we inferred that INPP4B might regulate GBC cells growth. To con rm our hypothesis, we chose GBC-SD and SGC996 cells for in vitro assay. Stable overexpression, knockdown and control of INPP4B in GBC-SD and SGC996 cells were established by different lentiviruses infection, respectively. Subsequently, we examined the effects of INPP4B on the growth and proliferation of GBC-SD and SGC996 cells using MTT and colonigenic assays. As shown in Fig. 2A, 2B and 2C, blocking the expression of endogenous INPP4B markedly inhibited the proliferation and colony formation of GBC-SD and SGC996 cells, whereas overexpression of INPP4B weakly promoted the proliferation and colony formation of these cells. In summary, our ndings suggest that blocking the expression of endogenous INPP4B has a greater effect on the proliferation of GBC cells than overexpression.

INPP4B regulates GBC cells apoptosis in vitro
Previous studies suggested that INPP4B is involved in the apoptosis of tumor cells [15,30]. The apoptosis levels of different lentivirus infection GBC-SD and SGC-996 cells were analysed by ow cytometry. Our results showed that both INPP4B overexpression and knockdown signi cantly increased the apoptosis rate of GBC-SD cell ( Fig. 3A and 3B). However, in SGC-996 cell, INPP4B overexpression markedly reduced the apoptosis rate, and INPP4B knockdown signi cantly increased the apoptosis rate ( Fig. 3C and 3D). Our results suggest that INPP4B regulates apoptosis in GBC cells, but the regulatory effects are different in different cell lines.

INPP4B promotes GBC cells migration and invasion in vitro
Scratch wound-healing assay and transwell assay were used to further investigate the effect of INPP4B on the migration and invasion ability of GBC cells. Scratch wound-healing assay con rmed that INPP4B overexpression markedly increased GBC-SD and SGC996 cells migration ability, while INPP4B knockdown had an opposite effect on the migration ability of these cells (Fig. 4A and 4B). Consistent with this results, transwell assays demonstrated that INPP4B overexpression notably increased GBC-SD and SGC996 cells invasion ability, while INPP4B knockdown had an opposite effect on the invasion ability of these cells ( Fig. 5A and 5B). Taken together, These dates suggest that INPP4B promotes GBC cells metastasis and invasion ability in vitro.

Discussion
INPP4B, as a phosphoinositide phosphatase, has been reported to be low in expression, and plays a tumor suppressive role in human prostate cancer, breast cancer and ovarian cancers by negatively regulating PI3K-Akt signaling [10,11,13]. More recently, some unexpected ndings indicate that INPP4B is signi cantly upregulated, which plays an oncogene role in AML, melanoma and colon cancer by activating SGK3, and is associated with the patients prognosis [17-19,31,] [20]. However, the expression and clinical signi cance of INPP4B in human GBC and its biological function on GBC cells have not been studied.
In our present study, we rst revealed that INPP4B is highly expressed in GBC tissues compared with nontumor tissues and is associated with GBC patient prognosis in different histopathological differentiation group. When we investigated the correlation between INPP4B expression and clinicopathological parameters and clinical prognosis, we got some interesting ndings. Table 1 and Fig. 1B revealed that INPP4B protein expression was associated with histopathological differentiation, and INPP4B expression in high-moderate differentiation tissues was higher than that in low-undifferentiated tissues. When we did not stratify the relationship between INPP4B expression and GBC prognosis, survival analysis and Cox regression analysis showed that INPP4B was not associated with overall survival in GBC patients and was not an independent prognostic factor (shown in Table 2 and Fig. 1C). When we strati ed the relationship between INPP4B expression and GBC prognosis according to differentiation grade, and we further found that GBC patients with high INPP4B expression had a better prognosis in high-moderate differentiation group, but a worse prognosis in low-undifferentiated group, which played a contradictory role. These results  [10], and its overexpression can inhibit the migration, invasion and angiogenesis of prostate cancer cells [33]. While, INPP4B acts as an oncogene in AML cells and colon cancer cells. INPP4B promotes the growth of AML cells [17], and INPP4B silencing inhibits colon cancer cell proliferation and retards colon cancer xenograft growth [18]. In our study, we explored the function of INPP4B in two GBC cell lines (GBC-SD, SGC996) by proliferation, colony formation, apoptosis, migration and invasion. Our result shown that knockdown of INPP4B in GBC-SD cell and SGC996 cell signi cantly suppressed proliferation, colony formation, migration and invasion ability; by contrast, overexpression of INPP4B in these two GBC cells notably increased migration and invasion ability, but weakly promoted cell proliferation and colony formation at different level. These ndings suggest that the tumor suppressor gene INPP4B plays a potential carcinogenic role in GBC. When we analyzed the effect of INPP4B on the apoptosis of these two GBC cell lines, we got some interesting phenomena. Overexpression of INPP4B in SGC996 cell signi cantly reduced its apoptosis rate, while knockdown of INPP4B notably increased the apoptosis rate. But for GBC-SD cell, it was confusing that both overexpression and knockdown of INPP4B all increased the apoptosis rate, which may be due to the multiple complex carcinogenic signaling pathways of INPP4B on different cells, which needs further study. In addition, this also re ects the heterogeneity of the two cell lines used in our study, so more biological function tests should be performed to validate the results using more cell lines and provide evidence for targeted therapy in different patients. In conclusion, our study suggest that INPP4B could be a potential marker for diagnosis and drug targeted therapy of GBC.
In summary, we rst identi ed that INPP4B is upregulated in china GBC tissues by immunohistochemistry, and it plays a contradictory prognostic role in the progression of GBC patients with different histopathological differentiation. We found that GBC patients with high expression of INPP4B have a better prognosis in high-moderate differentiation patients but a worse prognosis in lowundifferentiated patients. In vitro cell experiment further con rmed that INPP4B may play an oncogene role in GBC cells. We found that INPP4B knockdown could inhibit proliferation and colony formation, decrease cell migration and invasion capability, and increase in the apoptosis rate of GBC-SD and SGC996 cells; while INPP4B overexpression has an opposite effect on the biological behaviors of GBC-SD and SGC996 cells, except that it also increases the apoptosis rate of GBC-SD cells. These ndings suggest that INPP4B may play an important role in the pathogenesis and development of GBC. However, this study has some limitations that cannot be ignored. First, The number of enrolled patients in this study was relatively small, and more cases could more accurately assess INPP4B expression and its relationship with prognosis in GBC. Second, the reason why both INPP4B overexpression and INPP4B knockdown all can increase the apoptosis rate of GBC-SD cells. Finally, this study only proves that INPP4B plays an important role in the development of GBC from clinical signi cance to cell function studies. We need to further explore the more accurate mechanism regulation of INPP4B in the progression of GBC, which can help us provide new methods for the clinical treatment of GBC. This is what we plan to study in future work.

Conclusions
In conclusion, our study is the rst to assess the clinical signi cant and function of INPP4B in GBC. Our results demonstrat that INPP4B is highly expressed in GBC tissues and is signi cantly associated with poor overall survival in low-undifferentiated GBC patients, and with better overall survival in highmoderate differentiated GBC patients. In addition, we nd that INPP4B can promote wound healing, migration, invasion and proliferation in vitro, which suggests that INPP4B may be a potential therapeutic target for GBC patients. Further research will focus on the mechanisms underlying the potential for targeting INPP4B in GBC treatment.