The Mechanisms And Functions Of Up-regulation Of LDHB And Down-regulation Of CRABP1 By Loss Of TSC1/TSC2 Complex

The receptor tyrosine kinase (RTK)-phosphatidylinositol 3-kinase (PI3K)-AKT-mammalian target of rapamycin (mTOR) pathway plays a central role in regulating cell growth, proliferation, differentiation and survival. Components of this pathway include many proto-oncogenes, such as RTKs, PI3K, AKT and mTOR, and tumor suppressors, such as PTEN, TSC1 and TSC2. Due to gain-of-function mutations of proto-oncogenes and loss-of-function mutations of tumor suppressor, RTK-PI3K-AKT-mTOR signaling cascade is one of the most frequently activated signaling pathways in human cancers. However, the precise mechanisms downstream of the mTOR signaling leading to tumorigenesis remain largely unclear.In tumor cells, glucose is preferentially converted into lactic acid through aerobic glycolysis, which is known as the "Warburg effect". Lactate dehydrogenase (LDH), the key glycolytic enzyme catalyzing the formation of lactic acid from pyruvate, is often activated in many types of cancer. LDH is a tetrameric enzyme composed of combinations of two subunits (LDHA and LDHB). LDHA is elevated and activated in many cancers, and plays a crucial role in tumor initiation, maintenance and progression, whereas the significance of LDHB in tumor development remains more elusive and the regulation of LDHB expression is also less characterized. Over-expression of LDHB has been previously described in non-small cell lung cancer and testicular germ cell tumor, indicating that it may participate in tumor development.Vitamin A and its derivative, retinoic acid (retinoic acid, RA), play a wide range of roles in regulation of vertebrate development and cell differentiation. Vitamin A can be catalyzed to RA through a series of reactions. RA enters nucleus and binds to the retinoic acid receptors (RARs) and retinoid X receptors (RXRs), then RARs and RXRs form heterodimer and bind to the retinoic acid response element (RA-response element, RARE) of the targeted genes, thereby promoting gene transcription and producing a series of biological effects. Because of the function of RA in facilitating cell differentiation, inhibiting cell proliferation and inducing apoptosis, it has been used to treat some tumors, such as leukemia, melanoma and squamous cell carcinoma. Cellular retinoic acid binding protein 1 (Crabpl) plays an important role in regulating the function of RA by binding to RA in the cytoplasm and prevent it from entering the nucleus. The expression of Crabp1 is altered in many types of tumor, but the underlying mechanism remains unclear and the significance of these changes in tumor development has not yet completely understood.Since TSC1 and TSC2 protein complex is the major suppressor of mTOR signaling and mTOR hyperactivation is believed to be responsible for multiple organ benign tumor formation in tuberous sclerosis complex (TSC) disease due to inactive mutations of either TSC1 or TSC2, TSC1-or TSC2-null mouse embryonic fibroblasts (MEFs) have been widely used as a model for the study of mTOR signaling and related diseases. To investigate the effect of TSC2 deficiency, we compared the difference of the gene expression profiles between TSC2+/+and TSC2-/-MEFs using mRNA microarray assay. Among the differentially expressed genes, the mRNA abundance of LDHB is significantly increased while Crabp1 is dramatically down-regulated in TSC2-/-MEFs. We then explored the mechanism of the up-regulation of LDHB and the down-regulation of Crabp1 caused by loss of function of TSC1/TSC2 complex. We also investigated their biological functions in tumorigenesis.In the first part of this thesis, we report that the expression of LDHB, a critical enzymatic activator of glycolysis, was up-regulated in TSC2-/-MEFs, in an mTOR-dependent manner. The expression of LDHB gene was transactivated by signal transducer and activator of transcription 3 (STAT3), a known mTOR effector. Furthermore, mTOR enhanced LDHB expression through activation of STAT3. Reduction of LDHB blunted the tumorigenic potential of an oncogenic TSC2-null cell line in nude mice. We conclude that LDHB is a downstream target of mTOR and is important for oncogenic mTOR-mediated tumorigenesis. Hence LDHB may be targeted for the treatment of tumors caused by the aberrantly activated RTK-PI3K-AKT-mTOR signaling transduction cascade.In the second part of this thesis, we show that the loss of TSC1/TSC2 significantly reduced the expression Crabp1. Next, elevated DNMT1 by loss of TSC1/TSC2 was responsible for the promoter DNA methylation of Crabp1, leading to silence its expression. Furthermore, down-regulated Crabpl inhibited cell proliferation and promote cell differentiation. This result offers an alternative explanation for the benign nature of tumor in TSC patients. Finally, we provide data to show that down-regulation of Crabpl significantly enhanced the sensitivity of TSC1-/- or TSC2-/- MEFs to retinoic acid treatment. Synergistically inhibitory effect was achieved by treatment of TSC2 null MEFs with the combination of retinoic acid and rapamycin. Our findings may offer new therapeutic approaches for TSC and other TSC-related diseases.