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 findings indicate that INPP4B is significantly 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,]. To date, previous studies on the role of INPP4B in tumor suppressor gene and oncogene has been controversial, even in the same tumor. Guo et al. reported that INPP4B is frequently upregulated in human colon cancer tissues and cell lines and promotes tumorigenesis . Sung et al. and Ma et al. reported that INPP4B is down-regulated and have a tumor suppressor role in colorectal tumors . However, the expression and clinical significance of INPP4B in human GBC and its biological function on GBC cells have not been studied.
In our present study, we first revealed that INPP4B is highly expressed in GBC tissues compared with non-tumor 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 findings. 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 stratified 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 exhibited the dual role of INPP4B in different histopathological differentiation of GBC prognosis. This findings seem to be consistent with the report of Yang et al. Yang et al. study revealed that INPP4B plays a tumor suppressor role in non-metastatic colorectal cancer stem-like cells (CR-CSLCs) and plays an oncogene role in metastatic CR-CSLCs according to different mechanisms, although INPP4B is weakly expressed in non-metastatic CR-CSLCs and highly expressed in metastatic CR-CSLCs . These results suggest that even for the same tumor, INPP4B tends to promote tumor progression in more malignant tissues and cells, while inhibits tumor progression in relatively less malignant tissues and cells, regardless of its expression. Indeed, this phenomenon was also observed in another type of tumor we studied (date not shown). This may be one of the reasons that different researchers have come to different conclusions about tumor progression in INPP4B. Next, we further studied the function of INPP4B in GBC cells.
Previous studies on the role of INPP4B in tumor cells have shown that INPP4B has the function of tumor suppressor gene or oncogene in different tumor cells. INPP4B acts as a tumor suppressor gene in breast cancer cells and prostate cancer cells. Its knockdown can promote the proliferation and motility of breast cancer cells , and its overexpression can inhibit the migration, invasion and angiogenesis of prostate cancer cells . While, INPP4B acts as an oncogene in AML cells and colon cancer cells. INPP4B promotes the growth of AML cells , and INPP4B silencing inhibits colon cancer cell proliferation and retards colon cancer xenograft growth . 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 significantly 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 findings 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 significantly 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 reflects 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 first identified 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 low-undifferentiated patients. In vitro cell experiment further confirmed 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 findings 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 significance 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.