Identification Of Molecular Mechanisms Underlying Stemness And Metastasis Of Cancer Cells Promoted By The Lysine-specific Histone Demethylases 1

Hepatocellular carcinoma(HCC) and gastric cancer(GC) are two of the most common malignancies in China. Although recent studies have provided novel diagnostic biomarkers and therapeutic modalities, the overall outcome of patients with these cancers, especially of those with advanced diseases, are still dismal. Therefore, further investigations are needed to decipher molecular basis underlying development of liver cancer and gastric cancer, which may potentially benefit diagnosis and treatment of the cancers.It is increasingly clear that malignant growth of HCC is sustained by a subpopulation of cancer cells with stem-like properties, or Cancer stem cells(CSCs). Liver CSCs are endowed with self-renewal capacity and are supposed to be the “root" of malignant HCC. Elucidation of the mechanisms governing a self-renewing liver CSC shall be of important clinical relevance. Epigenetic modification of genomic DNA or histone proteins, such as histone methylation, is an important mechanism for the temporal and spatial control of gene regulation. Histone methylation plays an important role in the self-renewal, proliferation, and differentiation of normal stem cells, while abnormal expression of histone methylases occur in cancer development and many other disease settings as well. However, it is still largely unclear whether and how lysine-specific histone demethylases 1(LSD1) regulates self-renewal of liver CSCs. LSD1 is a flavin-containing amino oxidase(AO) that specifically catalyzes the demethylation of mono- and di-methylated lysine 4 at histone H3 and lysine 9 at H3 hrough a FAD(Flavin adenine dinucleotide)-dependent oxidative reaction. Previous studies have shown that LSD1 can maintain pluripotency of embryonic stem cells(ESC) and tumorigenicity of MLL-AF9 leukemia stem cells. Moreover, LSD1 is overexpressed in a variety of malignant tumor cells including HCC. These findings indicate that LSD1 may play an important role in regulating stem cell properties HCC cells.Invasion and metastasis of cancer cell are the major cause of death in cancer patients. Gastric cancer is characterized by high motility and invasion and distal metastasis. Therefore, it is of great interest to explore the mechanisms of invasion and metastasis of gastric carcinoma cells. Histone methylation is shown to regulate expression of genes associated with cancer invasion and metastasis. Consistently, LSD1 promotes tumor growth and metastasis in several types of cancer cells. For example, binding of LSD1 with the SNAG domain of Snail1 inhibits expression of E-cadherin and promotes epithelial mesenchymal transition(EMT). However, it is unclear how LSD1 regulates the invasion and metastasis of gastric cancer cells. Understanding of this regulatory mechanism of LSD1 will provide novel targets for intervention to inhibit cancer metastasis.Therefore, we investigated how LSD1 regulates the self-renewal in liver CSCs in the first part of the dissertation. In the second part, we studied the molecular mechanism of LSD1 function in promoting invasion and metastasis of gastric cancer cells. A list of experiment data and main conclusions are as following:Part One. LSD1-mediated maintenance of self-renewal of liver CSCs?. Expression of LSD1 in HCC and its clinical relevance.1. LSD1 is highly expressed in hepatocellular carcinoma cells, as compared to normal liver tissues.2. The levels of LSD1 expression in HCC tissues are positively associated with the histological grades and lymph node invasion of HCC.3. High levels of LSD1 expression in HCC tissues are predicative of shorter survival of patients??. Lgr5+ HCC cells exhibit CSCs properties and are chemoresistant.1. LSD1 expression is correlated with the expression of Lgr5.(1) Lgr5 expression in HCC cells is associated significantly with multiple lesions, lymph node invasion, higher histological grades(III-IV), and metastasis. Lgr5 expression is associated with shorter survival of patients with HCC.(2) The percentages of Lgr5+ cells in HCC lines are significantly higher than that in non-tumor hepatic L-O2 cells2. Lgr5+ HCC cells exhibit CSCs properties(1) Higher levels of stem cell related genes are detected in Lgr5+, but not Lgr5-HCC cells.(2) Upon induction of CSC differentiation, expression levels of Lgr5, Sox9, and Nanog are down-regulated, but expressions of Alb and Hnf-4 are up-regulated, which is indicative of hepatocytic differentiation of CSCs in vitro.(3) Lgr5+ HCC cells exhibit significantly stronger capacity to form tumorspheres than Lgr5- HCC cells in vitro.(4) Lgr5+ HCC cells are likely to be CSCs with potent drug resistance.(5) Lgr5+ HCC cells displayed enhanced tumorigenicity upon serial passages in vitro.???. LSD1 enhances CSCs properties in HCC cells.1. The expression level of LSD1 in Lgr5+ liver CSCs is significantly higher than that of Lgr5- cells;2. Higher levels of LSD1 are observed in Lgr5+, but not Lgr5- HCC cells.3. Induction of LSD1 overexpression expands the pool of Lgr5+ cells, and enhances CSC properties and drug resistance in HCC cells.4. Depletion of LSD1 attenuates the self-renewal of Lgr5+ CSCs and their drug resistance.?Ⅴ. Upregulation of LSD1 activates Wnt/β-catenin pathway to maintain stemness of Lgr5+ liver CSCs.1. LSD1 reduces the H3K4me1/2 methylation at the promoters of several repressors of β-catenin signaling to enhance β-catenin activity in Lgr5+ CSCs.2. The β-catenin signaling activity is crucial for LSD1 to regulate the tumorsphere formation and chemoresistance in Lgr5+ CICs.Ⅴ. Targeting LSD1 demethylase activity diminishes the activity of Lgr5+ CSCs.1. Treatment of LSD1 inhibitor increases the levels of total Prickle1 and APC in Lgr5+ CSCs.2. Treatment with LSD1 inhibitor suppresses the capacity of Lgr5+ CSCs to form tumorspheres in vitro.3. Treatment with LSD1 inhibitor attenuates drug resistance by Lgr5+ CSCs in vitro.4. Pharmacological ablation of LSD1 demethylase activity is of potential therapeutic value by attenuating the chemoresistance of Lgr5+ CSCs.Part Two. Regulation of invasion and metastasis of gastric cancer by LSD1?. Expression of LSD1 in GC tissues is associated with tumor malignancy.1. LSD1 is highly expressed in gastric cancer cells compared to normal tissues.2. The levels of LSD1 expression in gastric cancer tissues are positively associated with the histological grade, T stage, lymph node invasion, and TNM stage of gastric cancers.3. High level of LSD1 expression in gastric cancer tissues are correlated with shorter survivals of patients.??. LSD1 promotes the invasion and metastasis of gastric cancer cells.1. Knockdown of LSD1 expression inhibits the invasion and metastasis of gastric cancer cells.2. LSD1 inhibitors reduce the invasion and metastasis of gastric cancer cells.???. Molecular mechanisms underlying LSD1-associated invasion and metastasis of gastric cancer cells.1. Overexpression of LSD1 reduces the H3K4me1/2 level at the promoter region of WISP-2 gene to inhibit its transcription and protein expression, thereby activating TGF-β signaling pathway.2. Endogenous LSD1 suppresses WISP2 gene transcription and promotes endogenous TGF-β signaling activity.3. LSD1 suppresses WISP2 expression and thus sensitizes gastric cancer cells to TGF-β induced EMT.4. LSD1 promotes TGF-β-induced invasion and metastasis of gastric cancer cells.?Ⅴ. The expressions of LSD1 and WISP-2 are correlated in gastric cancer tissues1. The expression level of WISP-2 is reduced in gastric cancer cells.(1)WISP-2 staining is detected in the cytoplasm and on the cell membrane of gastric cancer cells. WISP-2 expression is reduced in gastric cancer tissues and metastatic lesions.(2)IHC staining of consecutive sections of gastric cancer tissues demonstrates that high expression of LSD1 is corrected with low expression of WISP-2 in gastric cancer cells.2. The expression of WISP-2 correlates with malignancy of gastric cancer. By analysis of 307 cases of gastric carcinoma, WISP-2 expression is negatively associated with higher histological grades(III-IV), T3/T4 stage, lymph node invasion, and metastasis.3. High expression of LSD1 is associated with low level of WISP-2 in gastric cancer.(1)WISP2 expression is associated significantly with longer periods of survival in patients with GC.(2)WISP2 expression is associated significantly with longer metastasis-free survival in patients with GC.(3)High expression of LSD1 is associated with low level of WISP-2 in gastric cancer.(4)High expression of LSD1 and low expression of WISP-2 collectively identify patients with much shorter survival time. Moreover, low expression of LSD1 and high expression of WISP-2 is associated with prolonged overall and metastasis-free survivals(5) Combined analyses of LSD1 and WISP-2 in gastric cancer tissues provide better prediction of metastasis probability of patients.In summary, the major findings of this study are as following:1. LSD1 plays an important role in the maintenance of self-renewal and tumorigenicity of cancer stem cells.2. By reducing H3K4me1/2 level at the promoter regions of Prickle1 and APC gene, LSD1 inhibits Prickle1 and APC gene expression, increases the activity of WNT signal pathway, and contributes to the maintenance of cancer stem cell self-renewal and tumorigenicity.3. LSD1 inhibitors can be used to as a sensitizer for chemotherapeutic agents.4. LSD1 promotes invasion and metastasis of gastric cancer cells.5. By reducing H3K4me1/2 level at the promoter region of WISP-2 gene, LSD1 activates TGF-β signaling pathway and promotes the invasion and metastasis of gastric cancer.We identify, for the first time, that LSD1 is essential for maintaining the stemness and chemo-resistance of lgr5+ liver cancer stem cells. LSD1 acts largely by reducing H3K4me1/2 levels at the promoters of the Prickle1 and APC genes to inhibit the expression of Prickle1 and APC thus activate the WNT signaling pathway. These findings suggest that LSD1 may be a potential molecular target for the treatment of liver cancer.We reveal a novel molecular mechanism of LSD1 in promoting gastric cancer invasion and metastasis. LSD1 reduces the H3K4me1/2 level in the promoter region of WISP-2 to inhibit WISP-2 expression, thereby activating TGF-β signaling pathway and promoting invasion and metastasis of gastric cancer cells. Therefore, the LSD1/WISP-2 signal axis may be potentially targetable to suppress invasiveness of gastric carcinoma.