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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

Tumor-derived IL-6 promotes OCCC tumor cell growth and survival(A) Top OCCC-specific genes in common between mouse and human tumors. Top six IPA and MSigDB (GSEA) predictions of the top upregulated genes are included. (B) RT-PCR validation of IL-6 expression in primary tumors and peritoneal metastases. Significant differences based on average normalized mRNA expression ±SD between peritoneal metastases or primary tumors and matched uninjected ovaries were calculated using a two-tailed Student’s t test. (C,D) Mouse IL-6 levels in the serum and ascitic fluid Arid1afl/fl;(Gt)Rosa26Pik3ca*H1047R mice, as measured by anti-IL-6 ELISAs. Significant differences based on the average protein concentration ±SD between wild-type versus Arid1afl/fl;(Gt)Rosa26Pik3ca*H1047R mice were calculated using a two-tailed Student’s t test. (E) IL-6 expression in an Arid1afl/fl;(Gt)Rosa26Pik3ca*H1047R tumor by IHC. (F) MTT absorbance values plotted with log antibody concentration (µg mL−1) for non-specific rat IgG-treated (control) or rat anti-mouse IL-6 treated Arid1afl/fl;(Gt)Rosa26Pik3ca*H1047R ascitic tumor cells after 96 hrs. of treatment. Plot represents the average absorbance value ±SD for treatment performed on three independent cell lines. Significant differences between control- and anti-IL-6-treated cells were calculated using a two-tailed Student’s t test (*significant p-value <0.05). (G) Primary ascitic tumor cells were treated 10, 1, 0.01, or 0 µg mL−1 rat anti-mouse IL-6 or non-specific Rat IgG-treated (control). (H,I) IL-6 expression in Arid1afl/fl;(Gt)Rosa26Pik3ca*H1047R ascitic tumor cells stably expressing control shRNAs or IL-6 shRNAs by ELISA and western blot. Each replicate represents in stable pool of IL-6 shRNA expressing cells from three independently isolated tumor cell lines. Significant differences based on the average protein concentration ±SD between control- versus IL-6 shRNA were calculated using a two-tailed Student’s t test. (J) MTT absorbance values plotted over time for control shRNA-, IL-6 shRNA-, or IL-6 shRNA-expressing cells supplemented with 10 ng/mL IL-6. Cells were plated at 2×10e4 cells per mL at hour 0, then MTT measurements were taken every 24 hours for a total of 96 hours. Significant differences between control- and IL-6-shRNA expressing cells were calculated using a two-tailed Student’s t test (*significant p-value < 0.05). (K,L) Whole-mount images of control- and IL-6-shRNA tumorgrafts (arrowheads) on the right flank of nude mice and images of corresponding H&E-stained tumorgraft sections. (M) Plot of growth rates for control- and IL-6-shRNA expressing tumorgrafts over 21 days of measurement. Significant differences between control- and IL-6-shRNA tumorgraft growth rates were calculated using a two-tailed Student’s t test. Only p-values <0.05 were considered significant.
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Figure 6: Tumor-derived IL-6 promotes OCCC tumor cell growth and survival(A) Top OCCC-specific genes in common between mouse and human tumors. Top six IPA and MSigDB (GSEA) predictions of the top upregulated genes are included. (B) RT-PCR validation of IL-6 expression in primary tumors and peritoneal metastases. Significant differences based on average normalized mRNA expression ±SD between peritoneal metastases or primary tumors and matched uninjected ovaries were calculated using a two-tailed Student’s t test. (C,D) Mouse IL-6 levels in the serum and ascitic fluid Arid1afl/fl;(Gt)Rosa26Pik3ca*H1047R mice, as measured by anti-IL-6 ELISAs. Significant differences based on the average protein concentration ±SD between wild-type versus Arid1afl/fl;(Gt)Rosa26Pik3ca*H1047R mice were calculated using a two-tailed Student’s t test. (E) IL-6 expression in an Arid1afl/fl;(Gt)Rosa26Pik3ca*H1047R tumor by IHC. (F) MTT absorbance values plotted with log antibody concentration (µg mL−1) for non-specific rat IgG-treated (control) or rat anti-mouse IL-6 treated Arid1afl/fl;(Gt)Rosa26Pik3ca*H1047R ascitic tumor cells after 96 hrs. of treatment. Plot represents the average absorbance value ±SD for treatment performed on three independent cell lines. Significant differences between control- and anti-IL-6-treated cells were calculated using a two-tailed Student’s t test (*significant p-value <0.05). (G) Primary ascitic tumor cells were treated 10, 1, 0.01, or 0 µg mL−1 rat anti-mouse IL-6 or non-specific Rat IgG-treated (control). (H,I) IL-6 expression in Arid1afl/fl;(Gt)Rosa26Pik3ca*H1047R ascitic tumor cells stably expressing control shRNAs or IL-6 shRNAs by ELISA and western blot. Each replicate represents in stable pool of IL-6 shRNA expressing cells from three independently isolated tumor cell lines. Significant differences based on the average protein concentration ±SD between control- versus IL-6 shRNA were calculated using a two-tailed Student’s t test. (J) MTT absorbance values plotted over time for control shRNA-, IL-6 shRNA-, or IL-6 shRNA-expressing cells supplemented with 10 ng/mL IL-6. Cells were plated at 2×10e4 cells per mL at hour 0, then MTT measurements were taken every 24 hours for a total of 96 hours. Significant differences between control- and IL-6-shRNA expressing cells were calculated using a two-tailed Student’s t test (*significant p-value < 0.05). (K,L) Whole-mount images of control- and IL-6-shRNA tumorgrafts (arrowheads) on the right flank of nude mice and images of corresponding H&E-stained tumorgraft sections. (M) Plot of growth rates for control- and IL-6-shRNA expressing tumorgrafts over 21 days of measurement. Significant differences between control- and IL-6-shRNA tumorgraft growth rates were calculated using a two-tailed Student’s t test. Only p-values <0.05 were considered significant.

Mentions: Since many of the upregulated genes we identified in cross-species comparisons with normal ovaries are likely to be found in other EOC subtypes (e.g. CA125) or generally found across most cancers, we next sought to identify an OCCC-specific gene signature for use in subtype-specific gene expression comparisons with the mouse tumors. To do this, we compiled a more refined ‘discriminant’ gene list for each subtype (e.g. clear-cell vs. all other EOC subtypes). We then compared the normalized raw expression values for each discriminant gene across all of the mouse tumor and human EOC samples. Using this analysis, we found that the gene expression patterns for mouse tumor and human OCCC samples were not statistically different (pval=0.6), suggesting that the OCCC-specific gene expression patterns are conserved between the two species (Fig. 5C). Several well-known markers of OCCC were identified as being highly expressed in the mouse tumors, including IL-6, STAT3, VCAN, and HIF1α, further supporting a role for heightened IL-6 signaling in OCCC tumor pathogenesis40, 41, 42 (Fig. 6A).


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)

Tumor-derived IL-6 promotes OCCC tumor cell growth and survival(A) Top OCCC-specific genes in common between mouse and human tumors. Top six IPA and MSigDB (GSEA) predictions of the top upregulated genes are included. (B) RT-PCR validation of IL-6 expression in primary tumors and peritoneal metastases. Significant differences based on average normalized mRNA expression ±SD between peritoneal metastases or primary tumors and matched uninjected ovaries were calculated using a two-tailed Student’s t test. (C,D) Mouse IL-6 levels in the serum and ascitic fluid Arid1afl/fl;(Gt)Rosa26Pik3ca*H1047R mice, as measured by anti-IL-6 ELISAs. Significant differences based on the average protein concentration ±SD between wild-type versus Arid1afl/fl;(Gt)Rosa26Pik3ca*H1047R mice were calculated using a two-tailed Student’s t test. (E) IL-6 expression in an Arid1afl/fl;(Gt)Rosa26Pik3ca*H1047R tumor by IHC. (F) MTT absorbance values plotted with log antibody concentration (µg mL−1) for non-specific rat IgG-treated (control) or rat anti-mouse IL-6 treated Arid1afl/fl;(Gt)Rosa26Pik3ca*H1047R ascitic tumor cells after 96 hrs. of treatment. Plot represents the average absorbance value ±SD for treatment performed on three independent cell lines. Significant differences between control- and anti-IL-6-treated cells were calculated using a two-tailed Student’s t test (*significant p-value <0.05). (G) Primary ascitic tumor cells were treated 10, 1, 0.01, or 0 µg mL−1 rat anti-mouse IL-6 or non-specific Rat IgG-treated (control). (H,I) IL-6 expression in Arid1afl/fl;(Gt)Rosa26Pik3ca*H1047R ascitic tumor cells stably expressing control shRNAs or IL-6 shRNAs by ELISA and western blot. Each replicate represents in stable pool of IL-6 shRNA expressing cells from three independently isolated tumor cell lines. Significant differences based on the average protein concentration ±SD between control- versus IL-6 shRNA were calculated using a two-tailed Student’s t test. (J) MTT absorbance values plotted over time for control shRNA-, IL-6 shRNA-, or IL-6 shRNA-expressing cells supplemented with 10 ng/mL IL-6. Cells were plated at 2×10e4 cells per mL at hour 0, then MTT measurements were taken every 24 hours for a total of 96 hours. Significant differences between control- and IL-6-shRNA expressing cells were calculated using a two-tailed Student’s t test (*significant p-value < 0.05). (K,L) Whole-mount images of control- and IL-6-shRNA tumorgrafts (arrowheads) on the right flank of nude mice and images of corresponding H&E-stained tumorgraft sections. (M) Plot of growth rates for control- and IL-6-shRNA expressing tumorgrafts over 21 days of measurement. Significant differences between control- and IL-6-shRNA tumorgraft growth rates were calculated using a two-tailed Student’s t test. Only p-values <0.05 were considered significant.
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Figure 6: Tumor-derived IL-6 promotes OCCC tumor cell growth and survival(A) Top OCCC-specific genes in common between mouse and human tumors. Top six IPA and MSigDB (GSEA) predictions of the top upregulated genes are included. (B) RT-PCR validation of IL-6 expression in primary tumors and peritoneal metastases. Significant differences based on average normalized mRNA expression ±SD between peritoneal metastases or primary tumors and matched uninjected ovaries were calculated using a two-tailed Student’s t test. (C,D) Mouse IL-6 levels in the serum and ascitic fluid Arid1afl/fl;(Gt)Rosa26Pik3ca*H1047R mice, as measured by anti-IL-6 ELISAs. Significant differences based on the average protein concentration ±SD between wild-type versus Arid1afl/fl;(Gt)Rosa26Pik3ca*H1047R mice were calculated using a two-tailed Student’s t test. (E) IL-6 expression in an Arid1afl/fl;(Gt)Rosa26Pik3ca*H1047R tumor by IHC. (F) MTT absorbance values plotted with log antibody concentration (µg mL−1) for non-specific rat IgG-treated (control) or rat anti-mouse IL-6 treated Arid1afl/fl;(Gt)Rosa26Pik3ca*H1047R ascitic tumor cells after 96 hrs. of treatment. Plot represents the average absorbance value ±SD for treatment performed on three independent cell lines. Significant differences between control- and anti-IL-6-treated cells were calculated using a two-tailed Student’s t test (*significant p-value <0.05). (G) Primary ascitic tumor cells were treated 10, 1, 0.01, or 0 µg mL−1 rat anti-mouse IL-6 or non-specific Rat IgG-treated (control). (H,I) IL-6 expression in Arid1afl/fl;(Gt)Rosa26Pik3ca*H1047R ascitic tumor cells stably expressing control shRNAs or IL-6 shRNAs by ELISA and western blot. Each replicate represents in stable pool of IL-6 shRNA expressing cells from three independently isolated tumor cell lines. Significant differences based on the average protein concentration ±SD between control- versus IL-6 shRNA were calculated using a two-tailed Student’s t test. (J) MTT absorbance values plotted over time for control shRNA-, IL-6 shRNA-, or IL-6 shRNA-expressing cells supplemented with 10 ng/mL IL-6. Cells were plated at 2×10e4 cells per mL at hour 0, then MTT measurements were taken every 24 hours for a total of 96 hours. Significant differences between control- and IL-6-shRNA expressing cells were calculated using a two-tailed Student’s t test (*significant p-value < 0.05). (K,L) Whole-mount images of control- and IL-6-shRNA tumorgrafts (arrowheads) on the right flank of nude mice and images of corresponding H&E-stained tumorgraft sections. (M) Plot of growth rates for control- and IL-6-shRNA expressing tumorgrafts over 21 days of measurement. Significant differences between control- and IL-6-shRNA tumorgraft growth rates were calculated using a two-tailed Student’s t test. Only p-values <0.05 were considered significant.
Mentions: Since many of the upregulated genes we identified in cross-species comparisons with normal ovaries are likely to be found in other EOC subtypes (e.g. CA125) or generally found across most cancers, we next sought to identify an OCCC-specific gene signature for use in subtype-specific gene expression comparisons with the mouse tumors. To do this, we compiled a more refined ‘discriminant’ gene list for each subtype (e.g. clear-cell vs. all other EOC subtypes). We then compared the normalized raw expression values for each discriminant gene across all of the mouse tumor and human EOC samples. Using this analysis, we found that the gene expression patterns for mouse tumor and human OCCC samples were not statistically different (pval=0.6), suggesting that the OCCC-specific gene expression patterns are conserved between the two species (Fig. 5C). Several well-known markers of OCCC were identified as being highly expressed in the mouse tumors, including IL-6, STAT3, VCAN, and HIF1α, further supporting a role for heightened IL-6 signaling in OCCC tumor pathogenesis40, 41, 42 (Fig. 6A).

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