Riassunto analitico
GNAS activating mutations are recurrent in around 2% of Colorectal cancer patients.
cAMP levels are reported to be low in several CRC cell lines, while other studies suggest that higher levels of cAMP result in lower malignant features. PDE4D is a cAMP selective phosphodiesterase that hydrolyze cAMP, thus inactivating its effect. Several papers report that PDE4D inhibition correlate with cAMP level enhancement, thus leading to reduced oncogenic features.
In order to study the potential roles played by GNAS activating mutation, cell lines edited by Horizon discovery (Waterbeach, England) were purchased with SW48 bearing GNAS R201H or KRAS G12D activating mutation, while HCT-116 bearing GNAS R201H activating mutation, respectively. Each of the mutated cell line was purchased with its respective parental cell line. This mutated GNAS subunit is consitutively activated, thus stimulating adenylate cyclase to produce more cAMP.
RNA-sequencing was performed, thus highlighting differential expression of genes and gene ontology pathway enrichment for genes up-regulated between each mutated cell line and the respective parental cell line.
In the SW48 KRAS mutated cell line was detected, from the RNA-seq data, a strong induction of DCLK1, which was further validated with qPCR and by western blotting.
No significant difference in adhesion was detected between any of the mutated cell lines compared to their parental ones.
No significant difference in the proliferation rate was detected between the two SW48 mutated cell line compared to their respective parental cell line.
HCT-116 GNASm cell line was significantly less proliferative and invasive than the parental one.
No difference in PKA activation, measured by western blotting with phospho PKA-substrate antibody, was detected. Moreover, HCT-116 basal cAMP level between mutated and parental cell line was not significantly different, although forskolin induction showed a much higher cAMP level enhancement in the HCT GNAS mutated cell line.
No significant difference in HCT-116 GNAS mutated cell line cAMP basal level can be explained by a strong induction of PDE4D, which was detected by the RNA-seq data and further validated with qPCR.
Similar PDE4D induction was validated in the SW48 GNAS mutated cell line.
HCT-116 proliferation assay with Ro (unspecific PDE4 inhibitor) and GEBR-7b (selective PDE4D inhibitor) treatment was performed, showing that the proliferation rate was lower only for the treated GNAS mutated cell line compared to the blank treatment with DMSO, while the HCT-116 parental cell line was insensitive to PDE4D inhibition. These results are in line with the fact that there is a strong expression of PDE4D only in the GNAS mutated cell line, and that PDE4D inhibition should lead to an increase of the cAMP level due to the activity of the constitutively activated GNAS subunit.
More experiments are needed to prove that PDE4D inhibition leads to a higher cAMP level enhancement and, that this is directly related to the decrease in the proliferation rate. Further functional assay with PDE4D inhibition, such as invasion and colony formation assay,could be performed to verify the reduction of other malignant features.
GNAS silencing with SiRNA could be performed in order to verify a direct role of the GNAS activated pathway in the strong up-regulation of PDE4D.
Then, pathway components down-regulation related to the cAMP/PDE4D signaling could be tested to better understand the mechanisms behind the phenotypic effect related to cAMP level enhancement.
Further studies about the GEBR-7b effect in Colorectal cancer are needed to better understand a potential tumor-suppressive effect.
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Abstract
GNAS activating mutations are recurrent in around 2% of Colorectal cancer patients.
cAMP levels are reported to be low in several CRC cell line, while other studies suggest that higher levels of cAMP result in lower malignant features. PDE4D is a cAMP selective phosphodiesterase that hydrolyze cAMP, thus inactivating its effect. Several papers report that PDE4D inhibition correlates with cAMP level enhancement, thus leading to reduced oncogenic features.
In order to study the potential roles played by GNAS activating mutation, cell lines edited by Horizon discovery (Waterbeach, England) were purchased with SW48 bearing GNAS R201H or KRAS G12D activating mutation, while HCT-116 bearing GNAS R201H activating mutation, respectively. Each of the mutated cell line was purchased with its respective parental cell line. This mutated GNAS subunit is constitutively activated, thus stimulating adenylate cyclase to produce more cAMP.
RNA-sequencing was performed, thus highlighting differential expression of genes and gene ontology pathway enrichment for genes up-regulated between each mutated cell line and the respective parental cell line.
In the SW48 KRAS mutated cell line was detected, from the RNA-seq data, a strong induction of DCLK1, which was further validated with qPCR and by western blotting.
No significant difference in adhesion was detected between any of the mutated cell lines compared to their parental ones.
No significant difference in the proliferation rate was detected between the two SW48 mutated cell line compared to their respective parental cell line.
HCT-116 GNASm cell line was significantly less proliferative and invasive than the parental one.
No difference in PKA activation, measured by western blotting with phospho PKA-substrate antibody, was detected. Moreover, HCT-116 basal cAMP level between mutated and parental cell line was not significantly different, although forskolin induction showed a much higher cAMP level enhancement in the HCT GNAS mutated cell line.
No significant difference in HCT-116 GNAS mutated cell line cAMP basal level can be explained by a strong induction of PDE4D, which was detected by the RNA-seq data and further validated with qPCR.
Similar PDE4D induction was validated in the SW48 GNAS mutated cell line.
HCT-116 proliferation assay with Ro (unspecific PDE4 inhibitor) and GEBR-7b (selective PDE4D inhibitor) treatment was performed, showing that the proliferation rate was lower only for the treated GNAS mutated cell line compared to the blank treatment with DMSO, while the HCT-116 parental cell line was insensitive to PDE4D inhibition. These results are in line with the fact that there is a strong expression of PDE4D only in the GNAS mutated cell line, and that PDE4D inhibition should lead to an increase of the cAMP level due to the activity of the constitutively activated GNAS subunit.
More experiments are needed to prove that PDE4D inhibition leads to a higher cAMP level enhancement and, that this is directly related to the decrease in the proliferation rate. Further functional assay with PDE4D inhibition, such as invasion and colony formation assay, could be performed to verify the reduction of other malignant features.
GNAS silencing with SiRNA could be performed in order to verify a direct role of the GNAS activated pathway in the strong up-regulation of PDE4D.
Then, pathway components down-regulation related to the cAMP/PDE4D signaling could be tested to better understand the mechanisms behind the phenotypic effect related to cAMP level enhancement.
Further studies about the GEBR-7b effect in Colorectal cancer are needed to better understand a potential tumor-suppressive effect.
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