Novel role for insulin as an autocrine growth factor for malignant brain tumour cells

Abstract

AT/RTs (atypical teratoid/rhabdoid tumours) of the CNS (central nervous system) are childhood malignancies associated with poor survival rates due to resistance to conventional treatments such as chemotherapy. We characterized a panel of human AT/RT and MRT (malignant rhabdoid tumour) cell lines for expression of RTKs (receptor tyrosine kinases) and their involvement in tumour growth and survival. When compared with normal brain tissue, AT/RT cell lines overexpressed the IR (insulin receptor) and the IGFIR (insulin-like growth factor-I receptor). Moreover, insulin was secreted by AT/RT cells grown in serum-free medium. Insulin potently activated Akt (also called protein kinase B) in AT/RT cells, as compared with other growth factors, such as epidermal growth factor. Pharmacological inhibitors, neutralizing antibodies, or RNAi (RNA interference) targeting the IR impaired the growth of AT/RT cell lines and induced apoptosis. Inhibitors of the PI3K (phosphoinositide 3-kinase)/Akt pathway also impaired basal and insulin-stimulated AT/RT cell proliferation. Experiments using RNAi and isoform-specific pharmacological inhibitors established a key role for the class I(A) PI3K p110alpha isoform in AT/RT cell growth and insulin signalling. Taken together, our results reveal a novel role for autocrine signalling by insulin and the IR in growth and survival of malignant human CNS tumour cells via the PI3K/Akt pathway.

Figure 1. Expression of the IR and its downstream signalling components in MRT and AT/RT cells

(A) Equal amounts of lysates from adult brain (Ad), foetal brain (Fe), foetal cerebellum (Ce), or the cell lines indicated were analysed by Western blot for expression of the proteins indicated. The cell lines analysed were AS, LP, MON, STM (MRT), BT-12, BT-16 (AT/RT) and DAOY (medulloblastoma). (B) Equal amounts of lysates from the cell lines indicated were analysed by Western blot for expression of INI1.

Figure 2. Human AT/RT cells secrete insulin under serum-free conditions

(A) Serum-free supernatants from AT/RT cells were analysed after 5 days in culture by ELISA to detect insulin production. Results are the means with S.D. from a representative experiment (out of three) performed in triplicate. (B) Serum-free supernatants from BT-16 cells (upper panel) or purified recombinant human insulin (lower panel) were analysed by MS. (C) Expression of insulin (left) and the IRβ (centre) in BT-16 cells was investigated by immunofluorescence (×100 magnification). The detection was performed using secondary antibodies coupled with FITC (insulin) or Cy3 (IRβ). The right panel shows the merged pictures and the cell nucleus stained with DAPI (4′,6-diamidino-2-phenylindole). (D) The expression of PDX-1 was detected in MRT and AT/RT cells using RT–PCR. GAPDH expression was analysed in parallel as a loading control.

Figure 3. A pharmacological inhibitor or a neutralizing antibody targeting the IR impairs AT/RT cell proliferation

(A) AT/RT cells were incubated in the presence of increasing concentrations of the inhibitor NVP-AEW541 in a medium containing 10% (squares) or 0.5% (diamonds) FCS. Cell proliferation was determined after 72 h. Results are the means with S.D. from a representative experiment (out of three) performed with eight repetitions. (B) BT-16 cells were incubated in serum-free medium in the presence of neutralizing antibodies targeting either the IGFIR or the IR (10 μg/ml) and cell proliferation measured after 72 h. Results are the means with S.D. from three experiments performed in quadruplicate (*, P<0.05 by ANOVA test). (C) BT-16 cells were incubated with increasing concentrations of insulin in serum-free medium in the presence of LY294002 (10 μM, diamonds) or vehicle (squares). Cell proliferation was measured after 72 h. Results are the means with S.D. from a representative experiment (out of three) performed with eight repetitions. (D) BT-12 cells were incubated for 20 h in the presence of increasing concentrations of the inhibitor NVP-AEW541 and the induction of apoptosis assessed by Western blot for the active fragments of caspase 3 (20 and 17 kDa, arrows). The blots were reprobed for lamin B as a loading control.

Figure 4. Down-regulation of IR by siRNA treatment impairs AT/RT cell proliferation

(A, B) BT-12 or BT-16 AT/RT cells were transiently transfected with non-targeting siRNA (ctr), or siRNA targeting the IGFIR or the IR, alone or in combination, where indicated. Cell proliferation was measured after 72 h. Results are the means with S.D. from three experiments performed in quadruplicate (*, P<0.05 by ANOVA test). (C) BT-12 AT/RT cells were transiently transfected with siRNAs as indicated and expression of the IRβ and IGFIRβ was analysed by Western blot after 72 h.

Figure 5. Insulin activates the PI3K/Akt pathway in AT/RT cells

(A) Serum-starved AT/RT cells were stimulated with growth factors where indicated (40 ng/ml EGF; 50 ng/ml Ins; 20 ng/ml SCF) for 10 min, and activation of Akt and ERK was determined by Western blot with phospho-specific antibodies. Abbreviations: p-Akt, Ser⁴⁷³-phosphorylated Akt; p-ERK, Thr²⁰²/Tyr²⁰⁴-phosphorylated ERK. (B) Serum-starved BT-16 cells were pretreated with NVP-AEW541 (2.5 μM), stimulated with the growth factors indicated (50 ng/ml) for 10 min and activation of Akt and S6K1 was determined as above. Abbreviation: p-S6K1, Thr³⁸⁹-phosphorylated S6K. (C) Serum-starved AT/RT cells were stimulated with the growth factors indicated (10 min) and anti-phosphotyrosine (pY) immunoprecipitates were analysed by Western blots for the relevant receptors. (D) BT-12 (left panel) or BT-16 (right panel) cells were incubated in a medium containing 10% (closed bars) or 0.5% (open bars) FCS in the presence of the inhibitors indicated [LY294002 10 μM (LY); rapamycin 20 ng/ml (Rapa); PD98059 25 μM (PD); Akt inhibitor 20 μM (Akt Inh)], or vehicle (DMSO). Cell proliferation was determined after 72 h. Results are the means with S.D. from a representative experiment (out of three) performed with eight repetitions. (E) BT-12 (left panel) or BT-16 (right panel) cells were transiently transfected with empty vector, or constructs encoding activated (myrAktCA) or dominant-negative Akt (AktDN). Cell proliferation was determined after 72 h. Results are the means with S.D. from three experiments performed in quadruplicate (*, P<0.05 by ANOVA test).

Figure 6. The PI3K p110α isoform controls AT/RT cell proliferation and insulin signalling

(A) BT-16 cells were incubated with increasing concentrations of isoform-specific PI3K inhibitors [0.01 μM (light grey bars), 0.1 μM (medium grey bars), 1 μM (dark grey bars), 10 μM (closed bars) or vehicle (open bars)]. Cell proliferation was determined after 72 h. Results are the means with S.D. from a representative experiment (out of three) performed in quadruplicate. (B) Serum-starved BT-16 cells were pre-incubated with the p110α inhibitor YM024 (micromolar concentrations) and stimulated with growth factors (50 ng/ml insulin; 50 ng/ml IGF-I) where indicated for 10 min. Akt activation was determined by Western blot with phospho-specific antibodies. Abbreviation: p-Akt, Ser⁴⁷³-phosphorylated Akt. (C, D) BT-16 cells were transiently transfected with shRNA constructs targeting PI3K isoforms (PIK3CA: p110α; PIK3CB: p110β) or empty vector (pRS). (C) Cell proliferation was assessed after 72 h. Results are the means with S.D. from a representative experiment (out of three) performed with eight repetitions. (D) Protein down-regulation was determined by Western-blot analysis.

Figure 7. Ectopic expression of INI1 reduces the proliferation of AT/RT cells, without effect on IR expression

(A) BT-16 cells were stably transfected with empty vector (pcDNA) or HA-tagged INI1. Cell proliferation in a medium containing 0.5% (open bars) or 10% (closed bars) FCS was determined after 72 h. Results are the means with S.D. from a representative experiment (out of three) performed with eight repetitions. (B) Expression of HA-tagged INI1 in the transfected BT-16 cells was verified by Western-blot analysis. (C) Insulin mRNA expression in BT-16 cells transfected with empty vector (pcDNA) or HA-tagged INI1 was determined by RT–PCR. GAPDH was analysed in parallel as a control. (D) The expression of the IRβ and the IGFIRβ in BT-16 cells transfected with empty vector (pcDNA) or HA-tagged INI1 was determined by Western blot.