The Role And Mechanistic Studies Of GATA4-NF-?B Pathway In The Adaptation Of Colorectal Cancer Cells To Acidic Microenvironment

Malignant tumors threat the health of human,and solid tumor occupies a large proportion in malignancies(about 85%or more).Because tumor cells tend to undergo glycolysis,which produces lactic acids,and there are usually poor vascularization and blood perfusion in solid tumors,cancer cells are usually immersed in an acidic microenvironment.While acidic extracellular microenvironment is detrimental to normal cells,it has been shown to promote the development and malignancy of tumor cells.It was reported that if the cells are cultured in acidic medium,the invasiveness and metastatic capacity of the tumor cells are enhanced.Short-term culture of breast cancer cells with low pH(6.7)medium resulted in decreased proliferation rate,G1 arrest and increased autophagy.Our previous study has discovered that upon long-term culture(for 3 months or longer)under low pH(6.5)medium,colorectal cancer(CRC)cells that became adapted to the acidic environment acquire more distinct features of epithelial-mesenchymal transistion(EMT)and show reduced proliferation,lower rate of apoptosis,increased autophagy and lower level of reactive oxygen species(ROS).While it was shown that low level of ROS is very important for the survival of CRC cells under acidic environment.However,autophagy is not involved in maintaining low level of ROS in CRC-AA cells.It is known that p62,one of the autophagic substrates,can positively regulate the anti-oxidative stress regulatory factor NRF2 and negatively regulate the GATA4-NF-?B pathway.At the same time,NRF2 and NF-?B also antagonize each other.Is the low level of ROS in CRC-AA cells related to NRF2?Does NF-?B play an antioxidant role in helping CRC cells adapt to an acidic environment?This thesis addressed the above questions and produced the following results:1.NRF2 is downregulated in CRC-AAThe protein levels of NRF2 in CRC and CRC-AA cells were examined by Western Blotting.Compared with CRC cells,the expression of NRF2 was significantly down-regulated in CRC-AA cells.siRNA interference of ATG5 in CRC and CRC-AA cells resulted in an upregulation of p62,but not NRF2.The downregulation of NRF2 in CRC-AA cells suggests that NRF2-based antioxidant defense is probably dispensable for the survival of CRC-AA cells.2.NF-?B is upregulated in CRC-AA and is essential for the survival of CRC-AA in acidic microenvironmentThe immunofluorescence assay showed that p65 is predominantly localized in nucleus in CRC-AA as compared with CRC cells.The protein levels of p-p65 and p65 in CRC and CRC-AA cells were examined by Western Blotting.Compared with CRC cells,the expression of p-p65 was significantly up-regulated in CRC-AA cells.Real-time quantitative PCR showed that the expression of inflammatory cytokines was significantly higher in CRC-AA than that in CRC cells.Colony formation experiment showed that BAY 11-7082,an inhibitor of NF-?B,could significantly inhibit the colony-forming ability of CRC-AA,but the difference was not obvious in the CRC group.Apoptosis assay by annexin V-PI double staining followed by flow cytometry showed that the proportion of apoptotic cells was significantly higher in CRC-AA than in CRC cells when cells were treated with BAY11-7082 or p65 siRNA.These results suggest that CRC-AA cells rely on NF-?B more than CRC cells for survival.3.NF-?B depends on its antioxidant function to promote the survival of CRC-AA cellsCRC and CRC-AA cells were treated with the NF-?B inhibitor BAY11-7082 and flow cytometry was used to detect ROS.The results showed that ROS were increased in cells treated with NF-?B inhibitors.Similarly,the level of ROS in p65 siRNA-treated cells was increased in CRC and CRC-AA cells when p65 expression was interfered with siRNA While representative antioxidant genes,including SOD and SOD2,were upregulated in CRC-AA,their expression was attenuated by BAY11-7082.Anti-oxidant NAC(10mM)was found to significantly rescue apoptosis caused by inhibition of NF-?B function.4.GATA4 is upregulated in CRC-AA cellsPrevious study showed that GATA4 can upregulate NF-?B,but is subjected to p62-mediated autophagic degradation.Because p62 is reduced in CRC-AA,we speculated that GATA4-NF-?B pathway might be activated in and contribute to the survival of CRC-AA cells.We therefore examined the levels of GATA4 in CRC and CRC-AA cells by Western Blotting.The results showed that the expression of GATA4 was up-regulated in CRC-AA cells.Real-time quantitative PCR showed that the expression of inflammatory cytokines was significantly lower in CRC-AA cells treated with GATA4 siRNA.Apoptosis assay showed that GATA4 siRNA resulted in significant increase in the proportion of apoptosis in CRC-AA cells,but had little effect on CRC cells.Flow cytometry measurement of ROS levels showed that ROS were elevated in GATA4 siRNA-treated cells.Anti-oxidant NAC(10mM)pretreatment significantly abolished the difference in the induction of apoptosis in CRC and CRC-AA cells by GATA4 siRNA.These results indicated that GATA4 is upregulated in CRC-AA cells and probably acts upstream of NF-?B in sustaining the survival of CRC-AA cells.5.Secretory factors in CRC-AA cells have pro-survival effect on CRC cells under extracellular acidic stressColony forming assay was used to test whether soluble factors produced by CRC-AA cells possess pro-survival effect on CRC cells under acidic stress.The results showed that the CRC cells cultured in acidic media containing CRC-AA conditioned supernatant had increased cloning capacity than control cells,suggesting that NF-?B mediated production of soluble factors may also contribute to the pro-survival effect of NF-?B activation under acidic stressTogether,our work showed that GATA4-NF-?B pathway is upregulated in CRC-AA cells and is critical for the survival of CRC-AA.Its pro-survival effect relies on its antioxidant function.NF-?B mediated production of soluble factors may also contribute to the pro-survival effect.