Inositol pyrophosphates promote tumor growth and metastasis by antagonizing liver kinase B1

Abstract

The inositol pyrophosphates, molecular messengers containing an energetic pyrophosphate bond, impact a wide range of biologic processes. They are generated primarily by a family of three inositol hexakisphosphate kinases (IP6Ks), the principal product of which is diphosphoinositol pentakisphosphate (IP7). We report that IP6K2, via IP7 synthesis, is a major mediator of cancer cell migration and tumor metastasis in cell culture and in intact mice. IP6K2 acts by enhancing cell-matrix adhesion and decreasing cell-cell adhesion. This action is mediated by IP7-elicited nuclear sequestration and inactivation of the tumor suppressor liver kinase B1 (LKB1). Accordingly, inhibitors of IP6K2 offer promise in cancer therapy.

Keywords: IP6K; LKB1; cell-matrix adhesion; cell–cell adhesion; metastasis.

Fig. 1.

IP6K2 up-regulates cell-matrix adhesion and down-regulates cell–cell adhesion. (A) Morphology of wild-type and IP6K2^−/− HCT116 cells 22 and 44 h after seeding. (B) Percentage of cells spread at 22 h. (C) Cell-matrix adhesion force measured by the centrifugation assay 22 h after plating. (D) Immunostaining of vinculin and actin in wild-type and knockout cells. Cells were starved overnight, trypsinized, and then plated on gelatin (10 µg/mL)-coated plates for 1 h before staining. Arrowheads point to focal adhesions. (E) Levels of phospho-FAK and phospho-LKB1 determined by Western blot. Cells were in suspension on regular plates or on fibronectin-coated plates for 30 min before harvest. (F) Morphology of wild-type and IP6K2^−/− HCT116 cells grown in suspension. (G) Immunostaining of E-cadherin in wild-type and knockout cells 44 h after plating. (H) Levels of E-cadherin determined by Western blot 44 h after plating. (I) Morphology of wild-type or IP6K1, IP6K2 knockdown HCT116 cells 28 h after seeding. (J) Levels of phospho-FAK in IP6K1 or IP6K2 knockdown HCT116 cells, determined by Western blot. (K) Levels of E-cadherin and phospho-LKB1 in IP6K1 or IP6K2 knockdown HCT116 cells. Error bars represent means ± SD; n = 3 in triplicate (**P < 0.01, Student’s t test).

Fig. 2.

IP6K2 mediates cell migration, invasion, tumor cell growth, and metastasis. (A) Growth curve of wild-type and IP6K2 null cells. (B) Anchorage-independent growth in agarose. (C) Cell migration (Left) through transwell and invasion through Matrigel (Right). Cells that have migrated/invaded were stained with DAPI and imaged using a fluorescence microscope. (D) In vitro tumor sphere growth. (E) Subcutaneous xenograft from wild-type and IP6K2^−/− HCT116 cells. Implanted tumors were grown for 3 wk. (F) Tumor volume of orthotopic transplant from wild-type and IP6K2^−/− HCT116 cells. (G) Liver metastasis from orthotopic transplant. The H&E staining reveals metastatic spots. Error bars represent means ± SD; n = 3 in triplicate (**P < 0.01 and *P < 0.05, Student’s t test).

Fig. 3.

Catalytic activity of IP6K2 is required for its actions. (A) Wild-type but not catalytically dead (K222A) IP6K2 rescues the defect in cell spreading (i), cell–cell adhesion (ii), cell clustering (iii), focal adhesion formation (iv), and E-cadherin staining (v). (B) Wild-type but not catalytically dead (K222A) IP6K2 rescues the defect in cell adhesion force, anchorage-independent growth, transwell migration, Matrigel invasion, and in vitro tumor sphere formation. Data are normalized to values from mock-rescued (vector-only) cells. (C) Levels of phospho-FAK and phospho-LKB1 in the three rescued cell lines with/without fibronectin treatment. (D) Levels of E-cadherin in the three rescued cell lines. (E) Growth curve of xenograft tumors from the three rescued cell lines. (F) Percentage of orthotopic-transplanted mice with liver metastasis, s.c. metastasis, intestinal polyps, and bloody stool. (G) Typical image of orthotopic-transplanted mice. The one transplanted using wild-type IP6K2-rescued cells displays s.c. tumor metastasis. (H) Mice orthotopically transplanted using wild-type IP6K2-rescued cells display intestinal polyps. (I) Mice orthotopically transplanted with wild-type IP6K2-rescued cells have a higher percentage of liver metastasis and more metastatic spots. Error bars represent means ± SD; n = 3 in triplicate (**P < 0.01 and *P < 0.05, Student’s t test).

Fig. 4.

IP7 promotes nuclear localization of LKB1 by disrupting LKB1-HSP90 binding. (A) LKB1 knockdown rescues the defect in FAK phosphorylation in IP6K2 null cells. (B) Enhanced protein tyrosine phosphatase activity in IP6K2 null cells. (C) The general tyrosine phosphatase inhibitor pervanadate (50 µM, 30 min) augments FAK phosphorylation in IP6K2 null cells. (D) Immunostaining of LKB1 reveals IP6K2-dependent differential localization. (E) IP6K2 coexpresssion enhances nuclear localization of LKB1. Cyt, cytoplasmic; Nuc, nuclear. (F) Coimmunoprecipitation between Flag-LKB1 and GST-IP6K2 wild-type or kinase-dead mutant.