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Identification of a novel synthetic lethality of combined inhibition of hedgehog and PI3K signaling in rhabdomyosarcoma.

Graab U, Hahn H, Fulda S - Oncotarget (2015)

Bottom Line: First, GANT61/PI103 cotreatment increases mRNA and protein expression of NOXA and BMF, which is required for apoptosis, since knockdown of NOXA or BMF significantly reduces GANT61/PI103-induced apoptosis.Third, ectopic expression of BCL-2 or non-degradable phospho-mutant MCL-1 significantly rescue GANT61/PI103-triggered apoptosis.Thus, combined GLI1/2 and PI3K/mTOR inhibition represents a promising novel approach for synergistic apoptosis induction and tumor growth reduction with implications for new treatment strategies in RMS.

View Article: PubMed Central - PubMed

Affiliation: Institute for Experimental Cancer Research in Pediatrics, Goethe-University, Frankfurt, Germany.

ABSTRACT
We previously reported that aberrant HH pathway activation confers a poor prognosis in rhabdomyosarcoma (RMS). Searching for new treatment strategies we therefore targeted HH signaling. Here, we identify a novel synthetic lethality of concomitant inhibition of HH and PI3K/AKT/mTOR pathways in RMS by GLI1/2 inhibitor GANT61 and PI3K/mTOR inhibitor PI103. Synergistic drug interaction is confirmed by calculation of combination index (CI < 0.2). Similarly, genetic silencing of GLI1/2 significantly increases PI103-induced apoptosis. GANT61 and PI103 also synergize to induce apoptosis in cultured primary RMS cells emphasizing the clinical relevance of this combination. Importantly, GANT61/PI103 cotreatment suppresses clonogenic survival, three-dimensional sphere formation and tumor growth in an in vivo model of RMS. Mechanistic studies reveal that GANT61 and PI103 cooperate to trigger caspase-dependent apoptosis via the mitochondrial pathway, as demonstrated by several lines of evidence. First, GANT61/PI103 cotreatment increases mRNA and protein expression of NOXA and BMF, which is required for apoptosis, since knockdown of NOXA or BMF significantly reduces GANT61/PI103-induced apoptosis. Second, GANT61/PI103 cotreatment triggers BAK/BAX activation, which contributes to GANT61/PI103-mediated apoptosis, since knockdown of BAK provides protection. Third, ectopic expression of BCL-2 or non-degradable phospho-mutant MCL-1 significantly rescue GANT61/PI103-triggered apoptosis. Fourth, GANT61/PI103 cotreatment initiate activation of the caspase cascade via apoptosome-mediated cleavage of the initiator caspase-9, as indicated by changes in the cleavage pattern of caspases (e.g. accumulation of the caspase-9 p35 cleavage fragment) upon addition of the caspase inhibitor zVAD.fmk. Thus, combined GLI1/2 and PI3K/mTOR inhibition represents a promising novel approach for synergistic apoptosis induction and tumor growth reduction with implications for new treatment strategies in RMS.

No MeSH data available.


Related in: MedlinePlus

NOXA and BMF are required for GANT61/PI103-induced apoptosisRD, TE381.T, RMS13, RH30 and VJ cells were transfected with non-silencing siRNA (siControl) or siRNA targeting BMF and/or NOXA and treated for 48 hours with PI103 (1 μM) and GANT61 (RD 6 μM; TE381.T 8 μM; RMS13 10 μM; RH30 8 μM; VJ 8 μM). Apoptosis was determined by DNA fragmentation of PI-stained nuclei using flow cytometry. Mean + S.D. of at least three independent experiments performed in triplicate are shown; *p < 0.05; **p < 0.01. Statistic analysis comparing combined knockdown to single knockdown is shown in Suppl. Table 2.
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Figure 4: NOXA and BMF are required for GANT61/PI103-induced apoptosisRD, TE381.T, RMS13, RH30 and VJ cells were transfected with non-silencing siRNA (siControl) or siRNA targeting BMF and/or NOXA and treated for 48 hours with PI103 (1 μM) and GANT61 (RD 6 μM; TE381.T 8 μM; RMS13 10 μM; RH30 8 μM; VJ 8 μM). Apoptosis was determined by DNA fragmentation of PI-stained nuclei using flow cytometry. Mean + S.D. of at least three independent experiments performed in triplicate are shown; *p < 0.05; **p < 0.01. Statistic analysis comparing combined knockdown to single knockdown is shown in Suppl. Table 2.

Mentions: To investigate the functional involvement of NOXA and BMF in apoptosis induction we knocked down these proteins by siRNA (Fig. S6). NOXA silencing significantly reduced GANT61/PI103-induced apoptosis in all cell lines and BMF knockdown significantly decreased apoptosis in all but RH30 cells (Fig. 4A). Simultaneous knockdown of both NOXA and BMF led to a further significant reduction of GANT61/PI103-induced apoptosis in RMS13 cells (Suppl. Table S2). These findings demonstrate that NOXA and BMF contribute to GANT61/PI103-induced apoptosis.


Identification of a novel synthetic lethality of combined inhibition of hedgehog and PI3K signaling in rhabdomyosarcoma.

Graab U, Hahn H, Fulda S - Oncotarget (2015)

NOXA and BMF are required for GANT61/PI103-induced apoptosisRD, TE381.T, RMS13, RH30 and VJ cells were transfected with non-silencing siRNA (siControl) or siRNA targeting BMF and/or NOXA and treated for 48 hours with PI103 (1 μM) and GANT61 (RD 6 μM; TE381.T 8 μM; RMS13 10 μM; RH30 8 μM; VJ 8 μM). Apoptosis was determined by DNA fragmentation of PI-stained nuclei using flow cytometry. Mean + S.D. of at least three independent experiments performed in triplicate are shown; *p < 0.05; **p < 0.01. Statistic analysis comparing combined knockdown to single knockdown is shown in Suppl. Table 2.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496179&req=5

Figure 4: NOXA and BMF are required for GANT61/PI103-induced apoptosisRD, TE381.T, RMS13, RH30 and VJ cells were transfected with non-silencing siRNA (siControl) or siRNA targeting BMF and/or NOXA and treated for 48 hours with PI103 (1 μM) and GANT61 (RD 6 μM; TE381.T 8 μM; RMS13 10 μM; RH30 8 μM; VJ 8 μM). Apoptosis was determined by DNA fragmentation of PI-stained nuclei using flow cytometry. Mean + S.D. of at least three independent experiments performed in triplicate are shown; *p < 0.05; **p < 0.01. Statistic analysis comparing combined knockdown to single knockdown is shown in Suppl. Table 2.
Mentions: To investigate the functional involvement of NOXA and BMF in apoptosis induction we knocked down these proteins by siRNA (Fig. S6). NOXA silencing significantly reduced GANT61/PI103-induced apoptosis in all cell lines and BMF knockdown significantly decreased apoptosis in all but RH30 cells (Fig. 4A). Simultaneous knockdown of both NOXA and BMF led to a further significant reduction of GANT61/PI103-induced apoptosis in RMS13 cells (Suppl. Table S2). These findings demonstrate that NOXA and BMF contribute to GANT61/PI103-induced apoptosis.

Bottom Line: First, GANT61/PI103 cotreatment increases mRNA and protein expression of NOXA and BMF, which is required for apoptosis, since knockdown of NOXA or BMF significantly reduces GANT61/PI103-induced apoptosis.Third, ectopic expression of BCL-2 or non-degradable phospho-mutant MCL-1 significantly rescue GANT61/PI103-triggered apoptosis.Thus, combined GLI1/2 and PI3K/mTOR inhibition represents a promising novel approach for synergistic apoptosis induction and tumor growth reduction with implications for new treatment strategies in RMS.

View Article: PubMed Central - PubMed

Affiliation: Institute for Experimental Cancer Research in Pediatrics, Goethe-University, Frankfurt, Germany.

ABSTRACT
We previously reported that aberrant HH pathway activation confers a poor prognosis in rhabdomyosarcoma (RMS). Searching for new treatment strategies we therefore targeted HH signaling. Here, we identify a novel synthetic lethality of concomitant inhibition of HH and PI3K/AKT/mTOR pathways in RMS by GLI1/2 inhibitor GANT61 and PI3K/mTOR inhibitor PI103. Synergistic drug interaction is confirmed by calculation of combination index (CI < 0.2). Similarly, genetic silencing of GLI1/2 significantly increases PI103-induced apoptosis. GANT61 and PI103 also synergize to induce apoptosis in cultured primary RMS cells emphasizing the clinical relevance of this combination. Importantly, GANT61/PI103 cotreatment suppresses clonogenic survival, three-dimensional sphere formation and tumor growth in an in vivo model of RMS. Mechanistic studies reveal that GANT61 and PI103 cooperate to trigger caspase-dependent apoptosis via the mitochondrial pathway, as demonstrated by several lines of evidence. First, GANT61/PI103 cotreatment increases mRNA and protein expression of NOXA and BMF, which is required for apoptosis, since knockdown of NOXA or BMF significantly reduces GANT61/PI103-induced apoptosis. Second, GANT61/PI103 cotreatment triggers BAK/BAX activation, which contributes to GANT61/PI103-mediated apoptosis, since knockdown of BAK provides protection. Third, ectopic expression of BCL-2 or non-degradable phospho-mutant MCL-1 significantly rescue GANT61/PI103-triggered apoptosis. Fourth, GANT61/PI103 cotreatment initiate activation of the caspase cascade via apoptosome-mediated cleavage of the initiator caspase-9, as indicated by changes in the cleavage pattern of caspases (e.g. accumulation of the caspase-9 p35 cleavage fragment) upon addition of the caspase inhibitor zVAD.fmk. Thus, combined GLI1/2 and PI3K/mTOR inhibition represents a promising novel approach for synergistic apoptosis induction and tumor growth reduction with implications for new treatment strategies in RMS.

No MeSH data available.


Related in: MedlinePlus