INPP4B restrains cell proliferation and metastasis via regulation of the PI3K/AKT/SGK pathway

Abstract Cervical cancer continues to be among the most frequent gynaecologic cancers worldwide. The phosphoinositide 3‐kinase (PI3K)/protein kinase B (AKT) pathway is constitutively activated in cervical cancer. Inositol polyphosphate 4‐phosphatase type II (INPP4B) is a phosphoinositide phosphatase and considered a negative regulatory factor of the PI3K/AKT pathway. INPP4B has diverse roles in various tumours, but its role in cervical cancer is largely unknown. In this study, we investigated the role of INPP4B in cervical cancer. Overexpression of INPP4B in HeLa, SiHa and C33a cells inhibited cell proliferation, metastasis and invasiveness in CCK‐8, colony formation, anchorage‐independent growth in soft agar and Transwell assay. INPP4B reduced the expression of some essential proteins in the PI3K/AKT/SGK3 pathway including p‐AKT, p‐SGK3, p‐mTOR, phospho‐p70S6K and PDK1. In addition, overexpression of INPP4B decreased xenograft tumour growth in nude mice. Loss of INPP4B protein expression was found in more than 60% of human cervical carcinoma samples. In conclusion, INPP4B impedes the proliferation and invasiveness of cervical cancer cells by inhibiting the activation of two downstream molecules of the PI3K pathway, AKT and SGK3. INPP4B acts as a tumour suppressor in cervical cancer cells.

Interaction of PI (3,4)P2 with the pleckstrin homology (PH) domain of AKT is required for dimerization and full activation of AKT, 8 so like phosphatase and tensin homolog (PTEN), INPP4B may act as a tumour suppressor by antagonizing the PI3K/AKT signalling pathway. 9 However, recent reports demonstrated the controversial role of INPP4B in carcinogenesis in various tumours. INPP4B level was lower in follicular-like thyroid cancer tissues, melanocytic neoplasm, prostate cancer and breast cancer than in corresponding normal tissues, and knock-down of INPP4B activated AKT and increased the migratory, invasive and proliferative capacity in these cancer cells. [9][10][11][12] In contrast, INPP4B was found as an oncogenic regulator in colon cancer, acute myeloid leukaemia and in a subset of melanomas. [13][14][15] INPP4B promoted cell proliferation and chemical resistance in these cancer cells, mainly by activating serum-and glucocorticoidregulated kinase 3 (SGK3). 13,15 Therefore, the role of INPP4B needs to be explored in other cancer models for an overall picture of its function.
Our previous study demonstrated liver kinase B1 (LKB1) as an important tumour suppressor in cervical cancer. Overexpression of LKB1 inhibited HeLa cell proliferation and activated the AMPK pathway. 16 More importantly, overexpression of LKB1 up-regulated INPP4B and reduced p-AKT level, which suggested that LKB1 is involved in negative regulation of the PI3K/AKT pathway by INPP4B.
In this study, we further assessed the function of INPP4B in cer-

| Western blot assay
Total protein was extracted from cell lines using RIPA Lysis Buffer

| Cell proliferation, colony formation and anchorage-independent growth assays
For cell proliferation assay, cells were seeded onto 96-well plates at 3 9 10 3 cells per well. Cell proliferation was determined by use of the Cell Counting Kit-8 (CCK-8) (Dojindo, Japan) as the manufacturer recommended. For colony formation assay, cells were cultured on 6well plates at 500 cells per well for 10-14 days, and then colonies were counted after fixing and staining with methyl alcohol and 0.5% crystal violet solution, respectively. Anchorage-independent growth assays were performed as described. 17

| Cell migration and invasion assays
Transwell inserts (8 lm, 24-well format; Corning) and Matrigel (BD Bioscience)-coated Transwell inserts were used to measure cell migration and invasion. In total, 2 9 10 4 cervical cancer cells in 0.2 mL serum-free medium were added in the upper chamber of 24well plates, with 0.6 mL DMEM containing 20% foetal bovine serum added to the lower chamber. After incubation for 24 to 48 hour, non-invading cells were removed with use of a cotton swab, and the remaining cells were fixed, stained with 0.5% crystal violet solution and photographed in 5 random fields under a microscope.  All samples were collected from Qilu Hospital of Shandong University. Normal cervical tissues were from patients with chronic cervicitis or uterine fibroids who had undergone total hysterectomy. Patient consent was obtained for use of the tissue. Diagnosis was based on the World Health Organization Classification of Tumors.

| Immunohistochemistry (IHC)
IHC involved the streptavidin-peroxidase-biotin IHC method according to standard procedures as we previously described. 17 Slides were incubated with primary antibody for INPP4B (1:150; Cell Signaling) overnight at 4°C. All stained slides were observed by 2 independent investigators.    (Thr320) ( Figure 4D). To verify our results, we referenced the data for INPP4B mRNA expression in the Oncomine database. Two available databases, Biewenga and Scotto Cervix, had similar results as we found ( Figure 6J).   pre-clinical validation for many tumour types. 28,29 INPP4B may also be a potential candidate for adjuvant therapy of cervical cancer and other tumours that are activated in the PI3K pathway.