Identification of inositol polyphosphate 4-phosphatase type II as a novel tumor resistance biomarker in human laryngeal cancer HEp-2 cells

Abstract

Although tumor resistance remains a significant impediment to successful radiotherapy, associated regulatory markers and detailed molecular mechanisms underlying this phenomenon are not well defined. In this study, we identified inositol polyphosphate 4-phosphatase type II (INPP4B) as a novel marker of radioresistance by systematically analyzing Unigene libraries of laryngeal cancer. INPP4B was highly expressed in radioresistant laryngeal cancer cells and was induced by treatment with either radiation or anticancer drugs in various types of cancer cells. Ectopic INPP4B overexpression increased radioresistance and anticancer drug resistance by suppressing apoptosis in HEp-2 cells. Conversely, INPP4B depletion with small interfering RNA resensitized HEp-2 as well as A549 and H1299 cells to radiation- and anticancer drug-induced apoptosis. Furthermore, radiation-induced INPP4B expression was blocked by inhibition of extracellular signal-regulated kinase (ERK). INPP4B depletion significantly attenuated radiation-induced increases in Akt phosphorylation, indicating an association of INPP4B-mediated radioresistance with Akt survival signaling. Taken together, our data suggest that ERK-dependent induction of INPP4B triggers the development of a tumor-resistance phenotype via Akt signaling and identify INPP4B as a potentially important target molecule for resolving the radioresistance of cancer cells.

Figure 1. Identification of INPP4B as a radioresistance-related protein in HEp-2 cells. (A) Parental HEp-2 cells and redioresistant RR-HEp-2 and RR-#6 clonal variants were left untreated or treated with 10 Gy radiation for 24 h. Gene transcripts were detected by conventional RT-PCR. ERBB2 and Bcl-xl were used as positive controls; p21 was used as a negative control; and GAPDH was used as a loading control. (B) Parental HEp-2 cells and RR-HEp-2 and RR-#6 variants were treated with the indicated doses of radiation. After 14 d, the survival fraction was determined by clonogenic survival assay; results are presented as a survival curve (top). INPP4B protein levels were determined by western blotting using β-actin as a loading control (bottom). (C) Parental (CON), radiosensitive (RS-#7, -#9 and- #18) and radioresistant (RR-#3, -#6 and -#13) HEp-2 cells were left untreated (-) or treated (+) with 4 Gy radiation. After 14 d, colony formation was quantified using an automatic colony counter; results are presented as a survival curve (top). INPP4B protein levels were determined by western blotting using β-actin was used as a loading control (bottom). The data represent typical results or mean values with standard deviations (n = 3).

Figure 2. Induction of INPP4B expressionin cancer cells by treatment with radiation or anticancer drugs. (A) Parental HEp-2 cells (top) or parental HEp-2 and RR-HEp-2 and RR-#6 variants (bottom) were treated with 10 Gy radiation for the indicated times. INPP4B protein levels were determined by western blotting using β-actin was used as a loading control. (B) A549, H460, HCT116 and MCF7 cells were left untreated (-) or treated (+) with 10 Gy radiation for 24 h. INPP4B transcript levels were determined by quantitative PCR using GAPDH as an internal control. (C) HEp-2 cells were left untreated (CON) or treated with radiation (IR; 10 Gy), bleomycin (Bleo; 10 μM), cisplatin (Cis; 10 μM), etoposide (Eto; 5 μM), or doxorubicin (Doxo; 1 μM) for 24 h. INPP4A and INPP4B transcript levels were determined by quantitative PCR using GAPDH as an internal control. (D) Parental HEp-2 cells and RR-HEp-2 and RR-#6 variants were treated with 10 Gy radiation for 24 h. INPP4B, INPP4A and PTEN transcript levels were determined by quantitative PCR (top) or conventional RT-PCR (bottom). GAPDH was used as an internal control or loading control. The data represent typical results or mean values with standard deviations (n = 4).

Figure 3. ERK-dependent induction of INPP4B expression by radiation in HEp-2 cells. (A) HEp-2 cells were treated with 10 Gy radiation for the indicated times. Expression and phosphorylation levels of MAPK proteins were determined by western blotting. (B) HEp-2 cells were left untreated (-) or treated (+) with 10 Gy radiation in the absence (Vehicle) or presence of 10 μM PD98059 (PD), 10 μM SB203580 (SB), or SP600125 (SP) and then incubated for 24 h. INPP4B protein levels were determined by western blotting using β-actin as a loading control (top). INPP4B transcript levels were determined by conventional RT-PCR (middle) or quantitative PCR (bottom). GAPDH was used as a loading control or an internal control. (C) HEp-2 cells were transfected with control siRNA (siCON) or 100 nM ERK-1/2 siRNA (siERK) for 48 h and then left untreated (-) or treated (+) with 10 Gy radiation for an additional 24 h. INPP4B and ERK protein levels were determined by western blotting using β-actin as a loading control (top). INPP4B transcript levels were determined by conventional RT-PCR (middle) or quantitative PCR (bottom). GAPDH was used as a loading control or an internal control. The data represent typical results or mean values with standard deviations (n = 3).

Figure 4. INPP4B-dependent regulation of radiation sensitivity in HEp-2 cells. (A, B) Parental HEp-2 cells were transfected with an empty vector (CON) or Myc-tagged INPP4B expression vector (INPP4B) and then incubated for 24 h. The cells were treated with the indicated doses of radiation (A) or 10 Gy radiation (B). After 14 d, colony formation was quantified using an automatic colony counter; results are presented as a survival curve (A). After 48 h, cell viability was determined with a FACScan flow cytometer; data are presented as the percentage of PI-positive cells (B, left). Cell morphology was observed by light microscopy (B, middle). Levels of cleaved-PARP and INPP4B proteins were determined by western blotting using β-actin as a loading control (B, right). (C, D) RR-HEp-2 cells were transfected with control siRNA (siCON) or 100 nM INPP4B siRNA (siINPP4B) and then cultured for 48 h. The cells were treated with the indicated doses of radiation (C) or 10 Gy radiation (D). Cell survival (C), cell viability (D, left), cell morphology (D, middle) and western blotting (D, right) experiments were performed as in A and B. The data represent typical results or mean values with standard deviations (n = 4).

Figure 5. INPP4B-dependent regulation of anticancer drug sensitivity in stably transfected INPP4B-HEp-2 cells. (A, B) Control HEp-2 (CON) and stable INPP4B-HEp-2 cells (INPP4B) were treated with vehicle, 10 μM bleomycin, 5 μM etoposide, or 1 μM doxorubicin for 24 h. The expression levels of cleaved-PARP and Myc-tagged INPP4B were determined by western blotting using β-actin as a loading control (A, top). Cell death was determined with a FACScan flow cytometer; data are presented as the percentage of PI-positive cells (A, bottom). Cell morphology was observed by light microscopy (B). (C, D) INPP4B-HEp-2 cells were transfected with control siRNA (siCON) or 100 nM INPP4B siRNA (siINPP4B) for 48 h and then treated with vehicle or 1 μM doxorubicin for an additional 24 h. Western blotting (C, top), cell death (C, bottom) and cell morphology (D) experiments were performed as in A and B. (E) INPP4B-HEp-2 variants (INPP4B-#2, -#4 and -#6) were transfected with control siRNA (siCON) or 100 nM INPP4B siRNA (siINPP4B) for 48 h and then treated with vehicle or 1 μM doxorubicin for an additional 24 h. The expression levels of cleaved-PARP and Myc-tagged INPP4B were determined by western blotting using β-actin as a loading control. The data represent typical results or mean values with standard deviations (n = 4).

Figure 6. Induction of cell death by INPP4B depletion in lung cancer cells. (A) A549 and H1299 cells were transfected with control siRNA (siCON) or 100 nM INPP4B siRNA (siINPP4B) for 48 h. INPP4B protein levels were determined by western blotting using β-actin as a loading control (top). INPP4B mRNA levels were determined by quantitative PCR using GAPDH as an internal control (bottom). (B, C) A549 (B) and H1299 (C) cells were transfected with control siRNA (-) or 100 nM INPP4B siRNA (+) for 48 h and then left untreated (-) or treated (+) with 1 μM doxorubicin for 24 h or 10 Gy radiation for 48 h. The levels of cleaved-PARP were determined by western blotting using β-actin as a loading control (top and middle). Cell morphology was observed by light microscopy (bottom). The data represent typical results or mean values with standard deviations (n = 4).

Figure 7. Modulation of Akt activity by INPP4B expression in laryngeal and lung cancer cells. (A) Parental HEp-2 and radioresistant RR-HEp-2 cells were treated with 10 Gy radiation for the indicated times. Expression and phosphorylation levels of Akt and GSK3α/β were determined by western blotting using β-actin as a loading control. (B) RR-HEp-2 cells were transfected with control siRNA (siCON) or 100 nM INPP4B siRNA (siINPP4B) for 48 h and then left untreated or treated with 10 Gy radiation for an additional 6 h. Expression and phosphorylation levels of Akt and GSK3α/β were determined by western blotting using β-actin as a loading control. (C, D) Control HEp-2 (CON) and stably transfected INPP4B-HEp-2 (INPP4B) cells (C) or RR-HEp-2, A549 and H1299 cells (D) were transfected with control siRNA (siCON) or 100 nM INPP4B siRNA (siINPP4B) for 48 h. Expression and phophorylation levels of Akt, GSK3α/β and INPP4B were determined by western blotting using β-actin as a loading control. (E, F) RR-HEp-2 cells were transfected with control siRNA (siCON) or 100 nM INPP4B siRNA (siINPP4B) for 48 h and then left untreated or treated with 10 Gy radiation for an additional 6 h. Akt (E) or PP2A (F) was immunoprecipitated from each sample and the resulting immune complexes were used to measure PP2A phosphatase activity. The PP2A inhibitor okadaic acid was used as a positive control. The data represent typical results or mean values with standard deviations (n = 4).