Lipid phosphatase SHIP2 functions as oncogene in colorectal cancer by regulating PKB activation

Abstract

Colorectal cancer (CRC) is the second most common cause of cancer-related death, encouraging the search for novel therapeutic targets affecting tumor cell proliferation and migration. These cellular processes are under tight control of two opposing groups of enzymes; kinases and phosphatases. Aberrant activity of kinases is observed in many forms of cancer and as phosphatases counteract such "oncogenic" kinases, it is generally assumed that phosphatases function as tumor suppressors. However, emerging evidence suggests that the lipid phosphatase SH2-domain-containing 5 inositol phosphatase (SHIP2), encoded by the INPPL1 gene, may act as an oncogene. Just like the well-known tumor suppressor gene Phosphatase and Tensin Homolog (PTEN) it hydrolyses phosphatidylinositol (3,4,5) triphosphate (PI(3,4,5)P3). However, unlike PTEN, the reaction product is PI(3,4)P2, which is required for full activation of the downstream protein kinase B (PKB/Akt), suggesting that SHIP2, in contrast to PTEN, could have a tumor initiating role through PKB activation. In this work, we investigated the role of SHIP2 in colorectal cancer. We found that SHIP2 and INPPL1 expression is increased in colorectal cancer tissue in comparison to adjacent normal tissue, and this is correlated with decreased patient survival. Moreover, SHIP2 is more active in colorectal cancer tissue, suggesting that SHIP2 can induce oncogenesis in colonic epithelial cells. Furthermore, in vitro experiments performed on colorectal cancer cell lines shows an oncogenic role for SHIP2, by enhancing chemoresistance, cell migration, and cell invasion. Together, these data indicate that SHIP2 expression contributes to the malignant potential of colorectal cancer, providing a possible target in the fight against this devastating disease.

Keywords: SHIP2; colorectal cancer; migration; phosphatases; small molecule inhibitor.

Figure 1. INPPL1 mRNA and SHIP2 protein expression are increased in colorectal dysplasia and carcinoma as compared to non-dysplastic tissue

(A–C) Using publicly available gene expression data from Oncomine, INPPL1 expression was analyzed in carcinoma tissue compared to adjacent normal colon tissue. Shown are the significant upregulation of INPPL1 in CRC in datasets from Hong et al. (A), Kaiser et al. (B) and Skrzypczakand et al. (C). (D) Tissues of patients dysplasia (n = 14), colorectal cancer (CRC, n = 11), and controls (inactive ulcerative colitis, n = 8), were stained for SHIP2 by immunohistochemistry. Representative examples (10× and 40× magnification) are shown. (E, F) SHIP2 staining was scored for percentage of positive intestinal epithelial cells as well as intensity of staining. (**p > 0.01; ***p > 0.001).

Figure 2. SHIP2 expression in a large cohort of CRC patients is correlated to a worse patient survival

IHC analysis of SHIP2 on a tissue micro array (TMA) of colorectal cancer patients (n = 347) and healthy adjacent tissue (n = 206). (A) Representative stainings of tissue cores are shown, with liver tissue as positive control. (B) Using the previously published web-application “cut-off finder” we divided the patients in two groups based on percentage SHIP2-positive intestinal epithelial cells (High/Low). Kaplan meier curves for overall survival, disease free survival, and distant metastasis free survival reveal a significant correlation between high SHIP2 expression and these clinical parameters.

Figure 3. SHIP2 phosphatase activity is increased in CRC, and chemical SHIP2 activity inhibition results in CRC cells death

(A) SHIP2 phosphatase assay shows reliable measurement of SHIP2 activity in CRC cell lines. Recombinant SHIP2 served as positive control and data were normalized to bead-only control. As SHIP2 precipitations were done under bead-saturating conditions, SHIP2 activity levels are a representation of intrinsic SHIP2 activity rather than different amounts of precipitated SHIP2. Insert shows SHIP2 protein levels in the cell line lysates. (B) SHIP2 activity was quantified in freshly frozen cancer and normal adjacent colonic tissue (n = 8), showing increased phosphatase activity in CRC. (C, D) Treatment of HCT116 and CACO-2 colorectal cancer cell lines with two different SHIP2 activity inhibitors (K149 and K103) results in a dose-dependent cell death. Since CACO-2 cells seem to be more resistant to SHIP2 inhibition, other cell lines with PI3K-mutations were tested. No relationship between SHIP2 inhibition and PI3K mutational status exists (E).

Figure 4. SHIP2 inhibition interferes with cancer signaling pathways, and sensitizes to 5-fluorouracil (5-FU) treatment

(A, B) Treatment of CACO-2 and HCT116 cells with K149 results in decreased PKB phosphorylation, and increased pS6 phosphorylation. In contrast, control treatment with the PI3K inhibitor LY2940002 reduces both PKB phosphorylation and its downstream target pS6. ERK phosphorylation is affected by neither inhibitor. (C, D) 5-FU kills colorectal cancer cells in a dose dependent manner. In the presence of low concentrations of SHIP2 inhibitor, 5-FU-induced cell death is enhanced, in particular with low concentrations of 5-FU (*p < 0.05).

Figure 5. Stable knockdown of SHIP2 influences PKB signaling, without affecting cell proliferation

(A, B) Lentiviral transduction of SHIP2 shRNA resulted in 80% reduction of SHIP2 expression levels, and reduces PKB phosphorylation as shon by western blot analysis. (C, D) Using MTT assays, cell proliferation was assessed, and no effect of SHIP2 knockdown was observed.

Figure 6. Modulation of SHIP2 expression affects migration, invasion, and adhesion in colorectal cancer cells

(A) Cell migration was measured using a ring-barrier system. HCT116 cell migration on gelatin was tracked during 24 h, with locations being captured using time-lapse microscopy every 12min (x = start, line = cell track). (B, C) Quantification of migrated path indicates that the total migration and effective migration were significantly reduced in SHIP2 knockdown cells. (***p < 0.001). (D) Beads were coated with either HCT116 SHIP2 knockdown or control cells for 24 hours, and embedded in a collagen gel matrix. Cells were allowed to invade the collagen matrix, and pictures were taking at 0 h, 24 h, and 48 h (examples in E). The cell dispersion from the bead (arrow) into the collagen matrix was measured, and a trend towards reduced invasion was observed in SHIP2 knockdown cells. Data represents at least four beads. (p ≤ 0.05) (F, G) CRC cell adhesion was determined by MTT assay of adherent cells after indicated time points, with fibronectin (FN) coating serving as control. SHIP2 knockdown cells adhere more efficient to culture plates compared to non-target control.