Role Of NLR Family And DAMP Family Of CRT Molecules In Tumorigenesis And Its Molecular Mechanism

Part 1: Expression profile of NLRs in human colorectal cancer and the potencial role of NLRX1 in protection against colon tumorigenesisPurpose: Chronic inflammation has been found to affect all stages of colon tumorigenesis including initiation, progression, and metastasis. NLRs(nucleotide-binding domain leucine-rich repeat proteins or NOD-like receptors) have been showed to be important regulators of inflammation and immunity. A subgroup of NLRs and the innate immune receptor, AIM2(absent-in-melanoma 2), can induce the assembly of a large caspase-1 activating complex called the inflammasome. Other NLRs regulate key signaling pathways such as NF-κB and MAPK. Since inflammation is a central component of colorectal cancer(CRC), this work was undertaken to analyze NLR and AIM2 expression in human CRC by combining bioinformatics analysis and experimental verification using clinical tissue samples. Additional experiments analyzed the association of(i) gene expression and cancer staging, and(ii) gene expression among inflammasome components.Experimental Design: Ten public CRC datasets from the Oncomine® Platform were analyzed. Genes analyzed include NLRP1, NLRP3, NLRP6, NLRP12, NLRC3, NLRC4, NLRC5, NOD1, NOD2 and AIM2. Additionally, forty case-matched cancer samples and adjacent healthy control tissues isolated from a cohort of Chinese CRC patients were profiled. At last,we expand the role of the negative regulator, NLRX1 in controlling colon tumorigenesis in mice model.Results: 1)Three patterns of gene expression in CRC are shown. The expression of NLRC3, a checkpoint of inflammation, and the inflammasome components NLRP1, NLRP3, NLRX1, NLRC4 and AIM2 were reduced in CRC. NOD1 and NOD2 expression was increased in CRC, while NLRC5, NLRP6 and NLRP12 showed little difference compared to controls. 2)Reduced expression of NLRC3, NLRP3 and NLRX1 in CRC was verified in all available databases analyzed and confirmed with our patient cohort. 3)Nlrx1-/-mice were highly susceptible to colitis and colitis-associated colon cancer. Nlrx1-/-colons have higher number of polyps compared to controls and have elevated levels of NF-κB and MAPK activity and increased expression of IL-6 and STAT3 activity. Lost of NLRX1 expressions in the stromal compartment lead to higher levels of NF-κB and increased polyp formation in the colon. APCmin/+mice that also lack Nlrx1 have increased mortality and increased tumor burden at an earlier time point. Nlrx1-/-APCmin/+ colons have higher levels of NF-κB and MAPK activity, which leads to increased levels of STAT3 activity.Conclusions:These findings from animals model and human samples analysis all together suggest that Nlrx1 is providing protection against colon tumorigenesis.Furthermore, This report reveals the potential value of NLRs and AIM2 genes as biomarkers of CRC and cancer progression.Part 2:DAMPs and CRTin lung cancertumorigenesisPurpose: Damage-associated molecular patterns(DAMPs) are molecules that are secreted, released or surface exposed by dying, stressed or injured cells. DAMPs can function as either adjuvant or danger signals for the immune system. DAMPs such as surface-exposed calreticulin(CRT), secreted ATP and passively released high mobility group protein B1(HMGB1) are vital for the immunogenic cell death(ICD) of cancer cells. Calreticulin(CRT) is an endoplasmic reticulum luminal Ca2+-binding chaperone protein and one of DAMPs family. The translocation of CRT from ER to cell membrane and then cleaved to serum play an important roal inlung cancer tumorigenesis.Results: By immunizing mice with recombinant fragment(r CRT/39-272), 26 clones of monoclonal antibodies(m Abs) were generated and characterized. Based on these m Abs, a microplate chemi-luminescent enzyme immunoassay(CLEIA) system with a measured limit of detection of 0.09 ng/ml was developed. Using this CLEIA system, it was found that soluble CRT(s CRT) level in serum samples from 58 lung cancer patients was significantly higher than that from 40 healthy individuals(only 9 were detectable, P < 0.0001). Among them, serum s CRT in the small cell lung cancer was lower than that in adenocarcinoma(P = 0.0085), while both were lower than that in the squamous cell carcinoma(P = 0.013, P = 0.0012, respectively). Moreover, it was found that s CRT in sera from the patients after chemotherapy was higher than that from the patients without chemotherapy(P = 0.042). Further study by immunohistochemistry showed that CRT was also highly expressed in the cytoplasm and on the membrane of the lung cancer cells, while there was a trace amount of CRT expression in normal lung cells. Correspondingly, the expression level of CRT on lung cancer cell membrane was associated with the tumor pathological grade.At last,we foundp38 MAPK, ERK, JNK or AKT-PI3 K inhibitors SB203580,U0126,PD98059 or LY294002 and ER-stress inducer Tunicamycin or Thapsigargin can induce CRT upregulated on lung cancer cell line m RNA between 12-24hours, but Flow results shows the upregulation are not significant on cell membrane. BRAF V600 e inhibitor PLX4720 can induce CRT higher expression in cytoplasm within 1 hour,the MIF of CRT on cell membrane after different time point treatment could show the potential that CRT could translocate from cytoplasm to membrane.Conclusions: This study demonstrates that s CRT concentration in sera of lung cancer patients is higher than that in sera of healthy individuals, and CRT expression level on lung cancer cell membrane is associated with tumor pathological classification and grade. These findings suggest that CRT may be used as a biomarker in lung cancer prediction and diagnosis.