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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

GANT61/PI103 cotreatment increases NOXA and BMF expressionRD, TE381.T, RMS13, RH30 and VJ cells were treated with 1 μM PI103 and/or GANT61 (RD 6 μM; TE381.T 8 μM; RMS13 10 μM; RH30 8 μM; VJ 8 μM) for indicated times (A) or 24 hours (B) A, protein expression of BMF and NOXA was analyzed by Western blotting. B, mRNA levels of pro-apoptotic BCL-2 proteins were determined by qRT-PCR. Mean + S.D. of three independent experiments performed in triplicate (B) or representative blots (A) are shown; *p < 0.05; **p < 0.01 comparing treated to untreated cells.
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Figure 3: GANT61/PI103 cotreatment increases NOXA and BMF expressionRD, TE381.T, RMS13, RH30 and VJ cells were treated with 1 μM PI103 and/or GANT61 (RD 6 μM; TE381.T 8 μM; RMS13 10 μM; RH30 8 μM; VJ 8 μM) for indicated times (A) or 24 hours (B) A, protein expression of BMF and NOXA was analyzed by Western blotting. B, mRNA levels of pro-apoptotic BCL-2 proteins were determined by qRT-PCR. Mean + S.D. of three independent experiments performed in triplicate (B) or representative blots (A) are shown; *p < 0.05; **p < 0.01 comparing treated to untreated cells.

Mentions: Since the observed cleavage pattern of caspases points to engagement of the mitochondrial apoptotic pathway by GANT61/PI103 cotreatment, we analyzed the effects of GANT61 and PI103 on expression levels of pro- and antiapoptotic BCL-2 family proteins, which play an important role in regulating mitochondrial apoptosis. Interestingly, treatment with GANT61 alone or in combination with PI103 led to upregulation of the proapoptotic protein NOXA (Fig. 3A), which was accompanied by upregulation of NOXA mRNA by GANT61/PI103 cotreatment in RD and RH30 cells (Fig. 3B). In addition, treatment with PI103 alone or in combination with GANT61 caused a substantial upregulation of BMF mRNA and protein levels (Fig. 3A, 3B). Furthermore, GANT61/PI103 cotreatment increased expression of BIM and reduced MCL-1 protein levels in RH30 cells, whereas it had little effects on expression of BCL-2, BCL-XL, BAX and BAK (Fig. S5). This suggests that GANT61/PI103 cotreatment shifts the ratio of pro- and antiapoptotic BCL-2 proteins towards 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)

GANT61/PI103 cotreatment increases NOXA and BMF expressionRD, TE381.T, RMS13, RH30 and VJ cells were treated with 1 μM PI103 and/or GANT61 (RD 6 μM; TE381.T 8 μM; RMS13 10 μM; RH30 8 μM; VJ 8 μM) for indicated times (A) or 24 hours (B) A, protein expression of BMF and NOXA was analyzed by Western blotting. B, mRNA levels of pro-apoptotic BCL-2 proteins were determined by qRT-PCR. Mean + S.D. of three independent experiments performed in triplicate (B) or representative blots (A) are shown; *p < 0.05; **p < 0.01 comparing treated to untreated cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: GANT61/PI103 cotreatment increases NOXA and BMF expressionRD, TE381.T, RMS13, RH30 and VJ cells were treated with 1 μM PI103 and/or GANT61 (RD 6 μM; TE381.T 8 μM; RMS13 10 μM; RH30 8 μM; VJ 8 μM) for indicated times (A) or 24 hours (B) A, protein expression of BMF and NOXA was analyzed by Western blotting. B, mRNA levels of pro-apoptotic BCL-2 proteins were determined by qRT-PCR. Mean + S.D. of three independent experiments performed in triplicate (B) or representative blots (A) are shown; *p < 0.05; **p < 0.01 comparing treated to untreated cells.
Mentions: Since the observed cleavage pattern of caspases points to engagement of the mitochondrial apoptotic pathway by GANT61/PI103 cotreatment, we analyzed the effects of GANT61 and PI103 on expression levels of pro- and antiapoptotic BCL-2 family proteins, which play an important role in regulating mitochondrial apoptosis. Interestingly, treatment with GANT61 alone or in combination with PI103 led to upregulation of the proapoptotic protein NOXA (Fig. 3A), which was accompanied by upregulation of NOXA mRNA by GANT61/PI103 cotreatment in RD and RH30 cells (Fig. 3B). In addition, treatment with PI103 alone or in combination with GANT61 caused a substantial upregulation of BMF mRNA and protein levels (Fig. 3A, 3B). Furthermore, GANT61/PI103 cotreatment increased expression of BIM and reduced MCL-1 protein levels in RH30 cells, whereas it had little effects on expression of BCL-2, BCL-XL, BAX and BAK (Fig. S5). This suggests that GANT61/PI103 cotreatment shifts the ratio of pro- and antiapoptotic BCL-2 proteins towards 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