Molecular Mechanism Of LKB1 Inhibit MMP1 Expression And Lung Cancer Cell Invasion

Lung cancer is one of the serious threats to human health.Existing treatments of lung cancer include surgery,platinum-based chemotherapy,radiotherapy,and targeted therapy.Although in the past ten years the treatment of lung cancer had greatly developed,early lung cancer prone to transfer and nearly 78% of the patients with non-small cell lung cancer were diagnosed as phase III or IV in China.The 5-year survival rate of theses patients is very low,so the high transfer rate of lung cancer is one of the main factors that lead to its high mortality rate.Understanding the molecular mechanisms of lung cancer metastasis is very significant to improve the lung cancer survival rates.Cancer invasion and metastasis is a complex process.In the process of lung cancer development,the chromosome and genome of the cancer cells are unstable,and gene amplifications,deletions or mutations frequently occur.These structural changes might affect the expression or function of the genes.One of the most important changes in cancer development is the activation of oncogene and inactivation of tumor suppressor gene.In recent years,the role of tumor suppressor LKB1 in the malignant progression and metastasis of NSCLC has received mmore and more attentions from oncologists.LKB1(Liver Kinase B1),also known as STK11(serine/theronine protein Kinase 11),is a serine/threonine protein Kinase activity.In the majority of sporadic tumors,LKB1 mutations are rare.But in NSCLC,LKB1 mutation rate can be as high as 20% ～ 30%,as the third frequently mutant gene after p53 and Kras.LKB1 plays an important role in regulating cell energy metabolism,cell growth and cell polarity through the downstream kinases.AMPK(AMP-regulated protein kinase)is one of the important downstream kinases of LKB1 and is critical in regulating cell energy metabolism.In addition,LKB1 can also activate other AMPK related kinase,such as MARKs(microtuble affinity-regulating kinases),which is involved in the regulation of microtubule stability and cell polarity LKB1 plays an important role in lung cancer metastasis.In Kras driven mouse model of lung cancer,LKB1 loss significantly promote lung cancer lymph node and distant metastasis;Moreover,LKB1 mutations leads to cancer that has spread to the shorter incubation period and higher propensity.To evaluate the impact of LKB1 on the invasion of lung cancer cells,we detected the expression of LKB1 in a series of NSCLC cell lines and established isogenic LKB1 stable knockdown cell lines using LKB1 wild type H1299 cells.We confirmed that LKB1 m RNA and protein levels were significantly suppressed in H1299 cells that stably express LKB1 sh RNA.Transwell assay showed that compared with the control group,LKB1 knockdown enhanced the invasion of lung cancer cells.At present,the molecular mechanism by which LKB1 inhibits lung cancer metastasis is poorly understood.Previous studies show that the effect of inhibition of NEDD9,LOX and SRC is very limited and only partially reverses lung cancer metastasis caused by LKB1 loss so we speculate that there may be other molecules or signaling pathway that plays an important role in the process.In order to further study the underlying molecular mechanism,we performed microarray assay and compared the gene expression profiles between the H1299-p LKO.1 and H1299 LKB1 sh RNA cells.We found a series of important molecules associated with tumor invasion and metastasis,notably,the Matrix Metalloproteinase 1(MMP1),aroused our great interest.MMP1 m RNA level was almost 50 times higher than the control group.MMP1,also known as collagen enzyme,is one of the members of MMP family.MMP1 plays a key role in tumor invasion and metastasis by degrading the protein composition in ECM and destroying cell tumor histologic barriers.In a wide range of solid tumors including colorectal cancer,gastric cancer,lung cancer,breast cancer,ovarian cancer,prostate cancer,thyroid cancer and brain tumors the expression of MMP closely correlates with the invasion and metastasis of tumors.Therefore,we propose that MMP1 mighth act as a key molecule downstream of LKB1 to regulate lung cancer invasion.In this thesis,we use Real-time PCR and Western Blot to validate the microarray data.Consistently,we found that LKB1 knockdown led to an increase in the levels of MMP1 m RNA and protein.In addition,we found that overexpression of wildtype LKB1 in LKB1 mutant NSCLC cells repressed MMP1 levels,whereas kinase deficient LKB1 did not have obvious effct on MMP1 expression.Moreover,in the specimens of lung cancer patients,MMP1 conversely correlated with LKB1 levels.The results above show that LKB1 negatively regulates the expression of MMP1 negatively.LKB1 may inhibit lung cancer cell invasion via downregulating MMP1 levels.MARK is one of the important kinases downstream of LKB1.We found that pharmacological inhibition of MARK by a small molecule inhibitors,increased ERK phosphorylation and enhanced MMP1 expression,suggesting that MARK might act downstream of LKB1 to negative regulate MMP1 regulation.Taken together,using techniques of molecular biology and cell biology,we found that loss of LKB1 increses lung cancer cell invasion and upregulates the expression of MMP1,in addition,our study suggests that MARK acts downstream of LKB1 to negatively regulate MMP1 expression.Our work thus provides novel molecular insights into the role of LKB1 as a tumor suppressor in lung cancer and offers rationals for the molecular targeted therapy of lung cancer in the future.