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Coexistent ARID1A-PIK3CA mutations promote ovarian clear-cell tumorigenesis through pro-tumorigenic inflammatory cytokine signalling.

Chandler RL, Damrauer JS, Raab JR, Schisler JC, Wilkerson MD, Didion JP, Starmer J, Serber D, Yee D, Xiong J, Darr DB, Pardo-Manuel de Villena F, Kim WY, Magnuson T - Nat Commun (2015)

Bottom Line: We further show that ARID1A and PIK3CA mutations cooperate to promote tumour growth through sustained IL-6 overproduction.Our findings establish an epistatic relationship between SWI/SNF chromatin remodelling and PI3K pathway mutations in OCCC and demonstrate that these pathways converge on pro-tumorigenic cytokine signalling.We propose that ARID1A protects against inflammation-driven tumorigenesis.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA [2] Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.

ABSTRACT
Ovarian clear-cell carcinoma (OCCC) is an aggressive form of ovarian cancer with high ARID1A mutation rates. Here we present a mutant mouse model of OCCC. We find that ARID1A inactivation is not sufficient for tumour formation, but requires concurrent activation of the phosphoinositide 3-kinase catalytic subunit, PIK3CA. Remarkably, the mice develop highly penetrant tumours with OCCC-like histopathology, culminating in haemorrhagic ascites and a median survival period of 7.5 weeks. Therapeutic treatment with the pan-PI3K inhibitor, BKM120, prolongs mouse survival by inhibiting the tumour cell growth. Cross-species gene expression comparisons support a role for IL-6 inflammatory cytokine signalling in OCCC pathogenesis. We further show that ARID1A and PIK3CA mutations cooperate to promote tumour growth through sustained IL-6 overproduction. Our findings establish an epistatic relationship between SWI/SNF chromatin remodelling and PI3K pathway mutations in OCCC and demonstrate that these pathways converge on pro-tumorigenic cytokine signalling. We propose that ARID1A protects against inflammation-driven tumorigenesis.

No MeSH data available.


Related in: MedlinePlus

Coexistent ARID1A-PIK3CA mutations induce IL-6 expression in a cooperative manner(A) IL-6 mRNA expression in AdCRE-infected primary OSE cells carrying all allele combinations. (B) Normalized IL-6 ELISA measurements of AdCRE-infected primary OSE cells carrying all allele combinations. Significant differences based on the average normalized expression value ±SD among mutant allele combinations versus wildtype were calculated using a two-tailed Student’s t test. (C) Western blot showing ARID1A, P-AKT S473, total AKT, P-STAT3 Y705, and total STAT3 levels in AdCRE-infected primary OSE cells carrying all allele combinations. AdCRE-infected wildtype cells served as controls. (D) MTT cell viability assay on primary Arid1afl/fl;(Gt)Rosa26Pik3ca*H1047R OSE cells treated with AdControl, AdCRE, or AdCRE supplemented with 5 µg mL−1 anti-IL-6 antibodies. (E) Treatment of normal (wild type) primary OSE cells with vehicle (v/v), 10 ng mL−1 IL-6, or 10 ng mL−1 IL-6 supplemented with 5 µg mL−1 anti-IL-6 antibodies. Significant differences based on the average MTT absorbance value ±SD between three independent replicates of AdControl- versus AdCRE-infected or vehicle versus IL-6-treated cells were calculated using a two-tailed Student’s t test. (F) Western blot of normal primary OSE cells showing dose-dependent increases in P-STAT3 Y705 levels following IL-6 treatment. Co-treatment with 5 µg mL−1 anti-IL-6 antibodies blocked P-STAT3 Y705 induction. (G) ARID1A occupancy at the IL6 locus was detected by chromatin immunoprecipitation (ChIP) using anti-ARID1A antibodies (denoted as IP) on crosslinked chromatin from AdControl- or AdCRE-infected Arid1afl/fl;(Gt)Rosa26Pik3ca*H1047R primary OSE cells. Non-specific isotype matched antibodies (denoted as NS) were used in control ChIPs. ARID1A occupancy at the IL6 promoter (site or primer pair B) is reduced in ARID1A depleted cells treated with AdCRE. Average percent ChIP input ±SD represent data from experiments performed using three primary OSE cell isolations. Significant differences were calculated using a two-tailed Student’s t test. (H) Proposed model of IL-6 regulation by ARID1A and PIK3CA mutations in OCCC. Only p-values <0.05 were considered significant.
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Figure 7: Coexistent ARID1A-PIK3CA mutations induce IL-6 expression in a cooperative manner(A) IL-6 mRNA expression in AdCRE-infected primary OSE cells carrying all allele combinations. (B) Normalized IL-6 ELISA measurements of AdCRE-infected primary OSE cells carrying all allele combinations. Significant differences based on the average normalized expression value ±SD among mutant allele combinations versus wildtype were calculated using a two-tailed Student’s t test. (C) Western blot showing ARID1A, P-AKT S473, total AKT, P-STAT3 Y705, and total STAT3 levels in AdCRE-infected primary OSE cells carrying all allele combinations. AdCRE-infected wildtype cells served as controls. (D) MTT cell viability assay on primary Arid1afl/fl;(Gt)Rosa26Pik3ca*H1047R OSE cells treated with AdControl, AdCRE, or AdCRE supplemented with 5 µg mL−1 anti-IL-6 antibodies. (E) Treatment of normal (wild type) primary OSE cells with vehicle (v/v), 10 ng mL−1 IL-6, or 10 ng mL−1 IL-6 supplemented with 5 µg mL−1 anti-IL-6 antibodies. Significant differences based on the average MTT absorbance value ±SD between three independent replicates of AdControl- versus AdCRE-infected or vehicle versus IL-6-treated cells were calculated using a two-tailed Student’s t test. (F) Western blot of normal primary OSE cells showing dose-dependent increases in P-STAT3 Y705 levels following IL-6 treatment. Co-treatment with 5 µg mL−1 anti-IL-6 antibodies blocked P-STAT3 Y705 induction. (G) ARID1A occupancy at the IL6 locus was detected by chromatin immunoprecipitation (ChIP) using anti-ARID1A antibodies (denoted as IP) on crosslinked chromatin from AdControl- or AdCRE-infected Arid1afl/fl;(Gt)Rosa26Pik3ca*H1047R primary OSE cells. Non-specific isotype matched antibodies (denoted as NS) were used in control ChIPs. ARID1A occupancy at the IL6 promoter (site or primer pair B) is reduced in ARID1A depleted cells treated with AdCRE. Average percent ChIP input ±SD represent data from experiments performed using three primary OSE cell isolations. Significant differences were calculated using a two-tailed Student’s t test. (H) Proposed model of IL-6 regulation by ARID1A and PIK3CA mutations in OCCC. Only p-values <0.05 were considered significant.

Mentions: To determine if coexistent ARID1A-PIK3CA mutations are required for IL-6 induction, we utilized an AdCRE-inducible primary OSE cell culture model. IL-6 expression was measured following AdCRE-infection of primary OSE cells isolated from mice carrying all mutant allele combinations. Loss of ARID1A or PIK3CA activation alone led to significant increases in IL-6 expression by RT-PCR and ELISA, and IL-6 induction was further enhanced when ARID1A and PIK3CA were co-mutated together in Arid1afl/fl;(Gt)Rosa26Pik3ca*H1047R cells (Fig. 7A,B). Consistent with this, P-STAT3 Y705 levels also correlated with similar increases in IL-6 activity in AdCRE treated OSE cells (Fig. 7C). ARID1A loss in AdCRE-treated Arid1afl/fl or Arid1afl/fl;(Gt)Rosa26Pik3ca*H1047R OSE cells did not further enhance PI3K pathway activity (Fig. 7C). We found that Arid1afl/fl;(Gt)Rosa26Pik3ca*H1047R OSE cell proliferation was suppressed by treatment with anti-IL-6 neutralizing antibodies following mutation induction with AdCRE, and that recombiant IL-6 enhances normal OSE cell proliferation and IL-6-STAT3 signaling, further suggesting that IL-6 is both necessary and sufficient for tumor cell growth (Fig 7D–F). To determine if ARID1A is bound at the IL6 promoter under repressed conditions, we performed a series of ARID1A chromatin immunoprecipitation experiments in primary OSE cells. We used Arid1afl/fl;(Gt)Rosa26Pik3ca*H1047R OSE cells for these experiments so that AdCRE-treated or ARID1A-depleted cells could be used as a control for ARID1A immunoprecipitation specificity. In AdControl-treated cells, increased ARID1A occupancy was observed at sites near the IL6 promoter, but not at distal upstream or downstream sites (Fig. 7G). ARID1A occupancy at this site decreased in manner consistent with ARID1A depletion following AdCRE infection. These data indicate that coexistent ARID1A-PIK3CA mutations lead to IL-6 upregulation.


Coexistent ARID1A-PIK3CA mutations promote ovarian clear-cell tumorigenesis through pro-tumorigenic inflammatory cytokine signalling.

Chandler RL, Damrauer JS, Raab JR, Schisler JC, Wilkerson MD, Didion JP, Starmer J, Serber D, Yee D, Xiong J, Darr DB, Pardo-Manuel de Villena F, Kim WY, Magnuson T - Nat Commun (2015)

Coexistent ARID1A-PIK3CA mutations induce IL-6 expression in a cooperative manner(A) IL-6 mRNA expression in AdCRE-infected primary OSE cells carrying all allele combinations. (B) Normalized IL-6 ELISA measurements of AdCRE-infected primary OSE cells carrying all allele combinations. Significant differences based on the average normalized expression value ±SD among mutant allele combinations versus wildtype were calculated using a two-tailed Student’s t test. (C) Western blot showing ARID1A, P-AKT S473, total AKT, P-STAT3 Y705, and total STAT3 levels in AdCRE-infected primary OSE cells carrying all allele combinations. AdCRE-infected wildtype cells served as controls. (D) MTT cell viability assay on primary Arid1afl/fl;(Gt)Rosa26Pik3ca*H1047R OSE cells treated with AdControl, AdCRE, or AdCRE supplemented with 5 µg mL−1 anti-IL-6 antibodies. (E) Treatment of normal (wild type) primary OSE cells with vehicle (v/v), 10 ng mL−1 IL-6, or 10 ng mL−1 IL-6 supplemented with 5 µg mL−1 anti-IL-6 antibodies. Significant differences based on the average MTT absorbance value ±SD between three independent replicates of AdControl- versus AdCRE-infected or vehicle versus IL-6-treated cells were calculated using a two-tailed Student’s t test. (F) Western blot of normal primary OSE cells showing dose-dependent increases in P-STAT3 Y705 levels following IL-6 treatment. Co-treatment with 5 µg mL−1 anti-IL-6 antibodies blocked P-STAT3 Y705 induction. (G) ARID1A occupancy at the IL6 locus was detected by chromatin immunoprecipitation (ChIP) using anti-ARID1A antibodies (denoted as IP) on crosslinked chromatin from AdControl- or AdCRE-infected Arid1afl/fl;(Gt)Rosa26Pik3ca*H1047R primary OSE cells. Non-specific isotype matched antibodies (denoted as NS) were used in control ChIPs. ARID1A occupancy at the IL6 promoter (site or primer pair B) is reduced in ARID1A depleted cells treated with AdCRE. Average percent ChIP input ±SD represent data from experiments performed using three primary OSE cell isolations. Significant differences were calculated using a two-tailed Student’s t test. (H) Proposed model of IL-6 regulation by ARID1A and PIK3CA mutations in OCCC. Only p-values <0.05 were considered significant.
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Figure 7: Coexistent ARID1A-PIK3CA mutations induce IL-6 expression in a cooperative manner(A) IL-6 mRNA expression in AdCRE-infected primary OSE cells carrying all allele combinations. (B) Normalized IL-6 ELISA measurements of AdCRE-infected primary OSE cells carrying all allele combinations. Significant differences based on the average normalized expression value ±SD among mutant allele combinations versus wildtype were calculated using a two-tailed Student’s t test. (C) Western blot showing ARID1A, P-AKT S473, total AKT, P-STAT3 Y705, and total STAT3 levels in AdCRE-infected primary OSE cells carrying all allele combinations. AdCRE-infected wildtype cells served as controls. (D) MTT cell viability assay on primary Arid1afl/fl;(Gt)Rosa26Pik3ca*H1047R OSE cells treated with AdControl, AdCRE, or AdCRE supplemented with 5 µg mL−1 anti-IL-6 antibodies. (E) Treatment of normal (wild type) primary OSE cells with vehicle (v/v), 10 ng mL−1 IL-6, or 10 ng mL−1 IL-6 supplemented with 5 µg mL−1 anti-IL-6 antibodies. Significant differences based on the average MTT absorbance value ±SD between three independent replicates of AdControl- versus AdCRE-infected or vehicle versus IL-6-treated cells were calculated using a two-tailed Student’s t test. (F) Western blot of normal primary OSE cells showing dose-dependent increases in P-STAT3 Y705 levels following IL-6 treatment. Co-treatment with 5 µg mL−1 anti-IL-6 antibodies blocked P-STAT3 Y705 induction. (G) ARID1A occupancy at the IL6 locus was detected by chromatin immunoprecipitation (ChIP) using anti-ARID1A antibodies (denoted as IP) on crosslinked chromatin from AdControl- or AdCRE-infected Arid1afl/fl;(Gt)Rosa26Pik3ca*H1047R primary OSE cells. Non-specific isotype matched antibodies (denoted as NS) were used in control ChIPs. ARID1A occupancy at the IL6 promoter (site or primer pair B) is reduced in ARID1A depleted cells treated with AdCRE. Average percent ChIP input ±SD represent data from experiments performed using three primary OSE cell isolations. Significant differences were calculated using a two-tailed Student’s t test. (H) Proposed model of IL-6 regulation by ARID1A and PIK3CA mutations in OCCC. Only p-values <0.05 were considered significant.
Mentions: To determine if coexistent ARID1A-PIK3CA mutations are required for IL-6 induction, we utilized an AdCRE-inducible primary OSE cell culture model. IL-6 expression was measured following AdCRE-infection of primary OSE cells isolated from mice carrying all mutant allele combinations. Loss of ARID1A or PIK3CA activation alone led to significant increases in IL-6 expression by RT-PCR and ELISA, and IL-6 induction was further enhanced when ARID1A and PIK3CA were co-mutated together in Arid1afl/fl;(Gt)Rosa26Pik3ca*H1047R cells (Fig. 7A,B). Consistent with this, P-STAT3 Y705 levels also correlated with similar increases in IL-6 activity in AdCRE treated OSE cells (Fig. 7C). ARID1A loss in AdCRE-treated Arid1afl/fl or Arid1afl/fl;(Gt)Rosa26Pik3ca*H1047R OSE cells did not further enhance PI3K pathway activity (Fig. 7C). We found that Arid1afl/fl;(Gt)Rosa26Pik3ca*H1047R OSE cell proliferation was suppressed by treatment with anti-IL-6 neutralizing antibodies following mutation induction with AdCRE, and that recombiant IL-6 enhances normal OSE cell proliferation and IL-6-STAT3 signaling, further suggesting that IL-6 is both necessary and sufficient for tumor cell growth (Fig 7D–F). To determine if ARID1A is bound at the IL6 promoter under repressed conditions, we performed a series of ARID1A chromatin immunoprecipitation experiments in primary OSE cells. We used Arid1afl/fl;(Gt)Rosa26Pik3ca*H1047R OSE cells for these experiments so that AdCRE-treated or ARID1A-depleted cells could be used as a control for ARID1A immunoprecipitation specificity. In AdControl-treated cells, increased ARID1A occupancy was observed at sites near the IL6 promoter, but not at distal upstream or downstream sites (Fig. 7G). ARID1A occupancy at this site decreased in manner consistent with ARID1A depletion following AdCRE infection. These data indicate that coexistent ARID1A-PIK3CA mutations lead to IL-6 upregulation.

Bottom Line: We further show that ARID1A and PIK3CA mutations cooperate to promote tumour growth through sustained IL-6 overproduction.Our findings establish an epistatic relationship between SWI/SNF chromatin remodelling and PI3K pathway mutations in OCCC and demonstrate that these pathways converge on pro-tumorigenic cytokine signalling.We propose that ARID1A protects against inflammation-driven tumorigenesis.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA [2] Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.

ABSTRACT
Ovarian clear-cell carcinoma (OCCC) is an aggressive form of ovarian cancer with high ARID1A mutation rates. Here we present a mutant mouse model of OCCC. We find that ARID1A inactivation is not sufficient for tumour formation, but requires concurrent activation of the phosphoinositide 3-kinase catalytic subunit, PIK3CA. Remarkably, the mice develop highly penetrant tumours with OCCC-like histopathology, culminating in haemorrhagic ascites and a median survival period of 7.5 weeks. Therapeutic treatment with the pan-PI3K inhibitor, BKM120, prolongs mouse survival by inhibiting the tumour cell growth. Cross-species gene expression comparisons support a role for IL-6 inflammatory cytokine signalling in OCCC pathogenesis. We further show that ARID1A and PIK3CA mutations cooperate to promote tumour growth through sustained IL-6 overproduction. Our findings establish an epistatic relationship between SWI/SNF chromatin remodelling and PI3K pathway mutations in OCCC and demonstrate that these pathways converge on pro-tumorigenic cytokine signalling. We propose that ARID1A protects against inflammation-driven tumorigenesis.

No MeSH data available.


Related in: MedlinePlus