RACK1 Regulation Of Liver Cancer Chemotherapy Resistance And Its Interacting Protein CLEC-2 Function

PartⅠStudy on the role of RACK1 in the innate chemotherapy resistance of hepatocellular carcinomaHepatocellular carcinoma (HCC) is among the most common and lethal cancers in the human population, ranked the third most common cause of cancer-related death worldwide. Though routine surveillance can lead to early diagnosis and treatment when the tumor might be resectable, most HCC patients are diagnosed at advanced or late stages and could only receive palliative treatments, possibly due to the rapid progression of HCC. However, chemotherapy, serving as a common choice of palliative therapy, showed little benefit in the treatment of HCC patients. HCC generally displays inherent high resistance to chemotherapeutic drugs, and systemic or selective intra-arterial administration of any chemotherapy agent, which has marginal anti-tumor activity and shows no benefit for survival, is not recommended in clinical practice. At present, the underlying mechanism of the inherent high chemotherapy resistance of HCC remains unclear.In our study, we demonstrate that RACK1, the receptor for activated C-kinase 1, is highly expressed in normal liver and frequently up-regulated in HCC. Aberrant expression of RACK1 contributes to the chemotherapy resistance of HCC relying on its ribosome localization in vitro and in vivo, and the non-ribosome-binding mutant of RACK1 even sensitizes HCC cells to chemotherapy-induced apoptosis. Further study reveals that ribosome-associated RACK1 promotes the global protein synthesis, probably by acting on the eukaryotic initiation factor 4E (eIF4E). Ribosomal RACK1 directly associates with eIF4E in vitro and in vivo, and modulates the activity of eIF4E by recruiting PKCβⅡand promoting the phosphorylation of eIF4E on Ser 209. With the elevation of eIF4E activity, ribosomal RACK1 preferentially enhances translation of select mRNAs, many of which encode potent growth and survival factors, such as cyclin D1, c-myc, surviving and Bcl-2. This effect is also observed in vivo that the protein level of RACK1 positively correlates with the expression of cyclin D1, c-myc, surviving and Bcl-2. Translation suppression by CHX, or inhibiting the expression or activity of eIF4E, abolishes the anti-apoptotic effect of RACK1. We also observe that wild-type RACK1, but not its non-ribosome-binding mutant, promotes the formation of stress granules (SGs) upon the chemotherapeutic stress. Depletion of RACK1, or overexpression of the non-ribosome-binding mutant, even suppress the G3BP-induced SGs formation, suggesting that RACK1 and its ribosome localization are required for the assembly of SGs. Moreover, overexpression of RACK1 promotes the activation of AKT and ERK, and induces the proliferation of HCC cells. Our research first gain insight into the role of RACK1 in HCC, and provide clues to understanding the underlying mechanism of inherent chemotherapy resistance in HCC. PartⅡStudy on the role of RACK1 in the regulation of CLEC-2 expressionCLEC-2 was first identified as one member of non-classical C-type lectins by sequence similarity to C-type lectin-like molecules with immune functions. Human CLEC-2 is a typeⅡtransmembrane receptor with N-glycosylation, displaying a single extracellular C-type lectin-like domain (CTLD) connected to transmembrane region by a stalk and a non-classic immunoreceptor tyrosine-based activation motif (D-x-Y-x-x-L motif, ITAM) in its cytoplasmic tail. Recently, CLEC-2 has been demonstrated as a novel activating receptor that is likely to underlie platelet activation by the snake toxin Rhodocytin and endogenous ligand Podoplanin through the phosphorylation of ITAM and recruitment of Syk to initiate downstream signaling pathway. Additionally, CLEC-2 also co-operates with DC-SIGN to facilitate the capture of HIV-1 by platelets. Mouse CLEC-2 (mCLEC-2) shares high homology with human counterpart. We have reported two new alternatively spliced transcripts of mCLEC-2, the homologous dimerization of full-length mCLEC-2 and its cleavage into a soluble form. MCLEC-2 is also expressed on the surface of murine peripheral blood neutrophils, mediating internalization as well as the activation of neutrophils. So far, there is no report about the interacting partners with the cytoplasmic region of CLEC-2 except for Syk.In this study, by using the cytoplasmic region of human CLEC-2 (hCLEC-2) as bait, we perform a yeast two-hybrid screening in human leukemia cDNA library and identify the scaffold protein RACK1 as a potential interacting partner with hCLEC-2 in yeast. The direct interaction between RACK1 and hCLEC-2 is further identified by GST pull-down assay in vitro and co-immunoprecipation in vivo. Confocal analysis also reveals that RACK1 and hCLEC-2 co-localize in the cytoplasm of cells. Further research demonstrate that RACK1 decreases the stability of hCLEC-2 and inhibits the expression of both the unglycosylated and glycosylated forms of hCLEC-2. In vivo ubiquitination assay indicates that RACK1 promotes the proteosome-mediated degradation of hCLEC-2 by enhancing its ubiquitination. Proteosome inhibitors MG132 and lactacystin attenuate the degradation of CLEC-2 mediated by RACK1, while the treatment of choloquine, a lysosome inhibitor, shows little effect. Though RACK1 decreases the expression of glycosylated CLEC-2 in whole cell lysates, it does not impair the surface expression and signaling of CLEC-2. Taken together, these results suggest that RACK1 might be involved in the folding and protein quality control of CLEC-2, and provide clues to the understanding of the regulation of endogenous CLEC-2 expression. Part III Study on the recognition of CD74 by CLEC-2 and its functional effect on B-CLL leukemia cellsCLEC-2 is a type II transmembrane C-type lectin-like receptor, mainly distributed in liver, platelet, megakaryocyte, natural killer cells (NK cells) and antigen presentation cells (APC cells). However, surface expression CLEC-2 is only readily detected on platelet and megakaryocyte. In human platelet, CLEC-2 is detected as a doublet by western blot, with a major band migrating at 32kD and a minor band at 40kD. Recent studies demonstrate that CLEC-2 recognizes snake venom Rhodocytin as an exogenous ligand and Podoplanin as the endogenous ligand. In addition, CLEC-2 also co-operates with DC-SIGN to facilitate the capture of HIV-1 by platelets.In this study, to explore the potential endogenous ligand of CLEC-2 and gain insight into its biological function, we perform a yeast two-hybrid screening in human leukemia cDNA library by using the extracellular region of human CLEC-2 (hCLEC-2) as bait. We identify CD74 as a potential ligand for CLEC-2 in yeast. CD74, which is originally known as the MHC class II invariant chain, plays a critical role in the process of antigen presentation. Recent studies reveal that CD74 is also expressed on cell surface and functions as receptor to initiate downstream signaling. Importantly, CD74 is highly expressed on the surface of and mediated the proliferation of B chronic lymphocyte leukemia (B-CLL) cells. By expressing and purifying the recombinant protein of the extracellular part of CD74 fused to the Fc region of IgG1 (Fc-CD74), we identify in vitro that CD74 selectively binds to the 40kD form of hCLEC-2, but not its 32kD form. Confocal analysis also reveales that Fc-CD74 fusion protein co-localizes well with CLEC-2 on cell surface. We also transfect the hCLEC-2 construct into CHO cells (CHO/hCLEC-2), and co-culture them with Raji (B lymphoma cell line) cells. Results indicate that overexpression of CLEC-2 in CHO cells promotes the Raji adhesion, and enhances the activation of ERK in Raji cells. Blocking CD74 by using its antagonist antibody LN2 attenuates the ERK activation induced by CHO/hCLEC-2 cells. Cell cycle analysis and CFSE labeling assay demonstrate that co-culture with CHO/hCLEC-2 facilitates the S phase progression and proliferation of Raji cells in CD74-dependent manner. A megakaryocytic leukemia cell line, Dami, also promotes the proliferation of Raji cells through direct contact, while this effect could be blocked by using the CLEC-2 or CD74 monoclonal antibody. These results indicate that the 40kD form of CLEC-2 recognizes CD74 and promotes the initiation of its downstream signaling, and suggest that platelet may be involved in the progression and development of B-CLL by CLEC-2-CD74 interaction, thus providing CLEC-2 as a potential target for the treatment of B-CLL patients.