Colorectal Cancer Stem Cells Orchestrates Migration Through Regulating CLIC4and Prohibitin Connections

Colorectal cancer is the third malignant cancer worldwide. There have been1.2million newly dianognosed cases every year, and estimating more than60million patients died from colorectal cancer. Coloretal carcinoma is the second malignancy and the fourth leading death of cancer in China. Recent years, although the therapeutic methods of colorectal cancer has been greatly improved along with new technologies and chemotherapy drugs, the survival rate of patients has not significantly improved after surgery and postoperative chemotherapy. Recurrence and metastasis is the main cause of colorectal cancer, because more than half of colorectal cancer patients may be associated with micro-metastasis even before they have been underwent radical surgery. Therefore, it is urgent to find out key molecules to surpport evidences for interpreting mechanisms of colorectal cancer metastasis, so that the colorectal cancer patients would be curative by establishing a possible medical intervention to target specific molecules.Metastasis is a multi-step and complex process with deveolping secondary tumors in systemic organs. Cancer cells must detach from primary site, intravasate vessels, survive in circulation, adhere to endotheial cells of microcirculation in target organs, extravasate blood vessels or/and lymphatics, seed in the secondary sites, and then succeed in forming new tumors. Any part of the interruption in the process can not accomplish the metastases. Cancer stem cells, which is the tumor-initiating cells that the primary tumor and metastases formation must be rely on, are closely related to tumor metastasis. Directional migration of CSC is elaborately regulated by interaction of signal pathways and micro-environmental factors which promotes tumor cells expansion, spreading and metastasis. Therefore, aiming at the directional migration of CSC to inhibit tumor spreading and invasion and establish the biological shield system is a novel model for anti-metastasis.CSC is capable of self-renewal, differentiation, dormancy and resistance to chemoradiotherapy. Even after more than a decade of treatment, as long as the CSC is not effective to eradicate, the tumor will be relapse and metastasis. On one hand, the primary site of micro-environmental factors is suitable for CSC proliferation and differentiation; then the tumor expands by generating a great mount of progeny cells derived from CSC. On the other hand, CSC breaks into the blood vessels and lymphatics, seeking or inducing appropriate microenvironment, eventually reside in the target organ and produce progeny cells. This process can be finished in a short time or decades. In fact, there have been isolated cells with self-renewal and differentiation property as CSC from metastases. Some cells in metastatic tumors have the phenotypic characteristics as CSC. These indicated that the formation of metastases is also dependent on CSC. After predicting that there may be CSC with migration abilities, metastatic cancer stem cells (MCSC) have been identified in pancreatic cancer and colorectal cancer. Different subsets of CSC or different CSC subclones from the same cell lines have been proved to possess different metastatic potential, which indicated that CSC has the heterogeneity of metastatic potentials. Fighting against a given phenotype as targeting CSC is promising to eradicate malignant tumor. Eliminating the CSC with metastatic potential can restrain the tumor cells to stay at the primary site, and prevent malignant tumors from spreading and metastasis.We have obtained MCSC and non-metastatic cancer stem cells (non-MCSC) in colorectal cancer in previous study. After comparing the different phenotype between the two kinds of CSC, we have found that chloride intracellular channel protein4was associated with MCSC. Experiments in vitro and in vivo have demonstrated that CLIC4play roles on metastatic behaviors, especially indicating that CLIC4is related to cell migration. Here, we sought to screen candidate molecules and identify their interactions with CLIC4to highlight the molecular mechnism of the CSC migraion. It would be proposed a new method of breaking up the interaction between CLIC4and the candidate protein to block tumor metastasis.METHODS1. Screening proteins interacting with CLIC4in colorectal CSC(1) To screen CLIC4-interacting proteins by co-immunoprecipitation, mass spectrometry, bioinformatics methods and literature digging systems.(2) Using western blot and RT-PCR to detect the expression of the CLIC4-interacting proteins in SW480,17,40,17CSC and40CSC.(3) Using confocal laser scanning microscope to detect co-localization between CLIC4and its interacted proteins.2. Identification of the proteins interacting with CLIC4The results obtained by mass spectrometry were identified in vivo and in vitro by GST-pull down, co-immunoprecipitation and co-localization analysis.(1) GST-pull downProkaryotic expression vector of pGEX-4T-1-CLIC4and Pet32A-PHB were respectivly constructed. After the proteins were recommended to express in transformed cells of E. coli and then were purified, we have successfully obtained fusion proteins of CLIC4and PHB labeled with GST or His respectively. Then, the interaction role between CLIC4and PHB was validated by using GST-pull down technology in vitro.(2) Co-immunoprecipitation (CO-IP)Interactions between CLIC4and PHB were also verified from exogenous transfectants.Vectors carrying FLAG-CLIC4and HA-PHB were both constructed into293T cells. Co-immunoprecipitation was carried out respectively by agarose beads and western blots with anti-HA or anti-FLAG. It proposed to make sure whether exogenous CLIC4interacted with exogenous PHB.(3) Co-localization analysis293T cells were transfected together with PLVTHM-FLAG-CLIC4and PLVTHM-HA-PHB. After cultured for48hours, the cells were marked with fluorescence with anti-FLAG and anti-HA coming from different genus. Then, the cells were observed whether exogenous CLIC4and exogenous PHB could be identified with co-localization by confocal microscopy.Colorectal CSC was also identified by double immuno-labled to detect whether endogenic CLIC4and endogenic PHB could co-locate.(4) The interaction domains between CLIC4and PHBWe obtained the possible structural domains of CLIC4and PHB by using bioinformatics and literature analysis. Each of them was designed three mutants using immunoprecipitation technology (IP), and the interaction sites between CLIC4and PHB were obtained.3. Annotation of CLIC4and PHB(1) CLIC4and PHB were analyzed by immunohistochemistry in400cases of primary colorectal cancer with10years follow-up. The two proteins expression was anlysed in association with patient’s clinical characteristics, including gender, age, tumor stage, histopathological grading, metastasis and prognosis.(2) The lentiviral vector and pLVTHM-PHB vector were co-transfected into40CSC.40CSC for stable expression of PHB was obtained by limited dilution method and then it was confirmed by western blotting analysis.(3) The oligos of PHB siRNA were transfected transiently into17. Western blot and qRT-PCR were performed to detect the expression of PHB. The oligo with the most efficient interference upon PHB was packaged into HIV-1virus. Then, the virus was transfected into17CSC.17CSC for stable interference of PHB was obtained by limited dilution method and then it was confirmed by western blotting analysis.(4) We have investigated the effects of CLIC4and PHB overexpression and/or inteference on the biological behaviours of17CSC and40CSC by MTT assay, soft agar assay, invasive assay and migration assay in vitro.(5) Nude mice subcutaneous tumor forming assay and tail vein injection assay were performed to detect tumorigenicity and pulmonary dissemination capacity.4. The mechanism of colorectal cancer stem cells orchestrating migration through regulating CLIC4and PHB connections(1) The phenotypes of CSC was detected and confirmed by western blot in the interfered CLIC4and PHB in17CSC or overexpressed CLIC4and PHB in40CSC.(2) Proteins related to apoptosis was detected and confirmed by western blot in the interfered CLIC4and PHB in17CSC or overexpressed CLIC4and PHB in40CSC. (3) Proteins related to EMT was detected and confirmed by western blot in the interfered CLIC4and PHB in17CSC or overexpressed CLIC4and PHB in40CSC.(4) Using confocal laser scanning microscope to detect cytoskeletal proteins including F-actin and microtubule which induced by PHB.Results1. Screening proteins interacting with CLIC4in colorectal CSC(1) Firstly, we have screened CLIC4-interacting proteins including PHB, RPL7A, MACF1, SMARCA5, XRCC5.(2) Western blot and RT-PCR showed PHB, RPL7A, MACF1, SMARCA5and XRCC5were exepressed in SW480,17,40,17CSC and40CSC.(3) The results of confocal microscopy showed that PHB, RPL7A and MACF1can co-locate with CLIC4endogenously. SMARCA5can co-locate with CLIC4at mitotic stage. XRCC5can not co-locate with CLIC4. After bioinformatic and literature analysis, we selected PHB to perform the next experiments.2. Identified the protein interacting with CLIC4(1) We found that GST-CLIC4fusion protein could combine with PHB, but GST, the control, did not do that. Therefore, the result confirmed the interaction between CLIC4and PHB in vitro.(2) Interactions between CLIC4and PHB from exogenous transfectants were verified by co-immunoprecipitation.(3) Co-localization between CLIC4and PHB were verified by confocal microscopy from endogenous proteins. The Pearson’s coefficient was0.94.(4) The interaction sites of peptide domain were at67-100amino acids of CLIC4 and74-116amino acids of PHB, which was confirmed by co-immunoprecipitation.3. Annotation of CLIC4and PHB(1) IHC showed only a few cancer cells were positive for CLIC4and PHB, which is similar with the fraction of CSC in cancer cells as1/1000～1/100. The relationship between the CLIC4/PHB and clinicopathological data were analyzed after a10-year follow-up. CLIC4and PHB had no correlation with age, gender, differentiation or tumor size in the CRC patients (P>0.05). However, CLIC4was closely correlated with differentiation and the AJCC stages (P=0.001). CLIC4were significantly correlated with the overall survival of patients (P<0.05). PHB was losely associated with tumor location and vessel invasion (P=0.006, P=0.04).(2) PHB overexpression was successfully transfected into the target cells, which was detected and confirmed by western blot in the transfected CSCs or cell lines.(3) PHB interference was detected and confirmed by western blot in the transfected CSCs or cell line, which was revealed that PHB-homo-649was successfully interfered.(4) CLIC4overexpressed or PHB intefered cells showed that the proliferative rates significantly increased by compared with mock cells as determined in MTT assay (P<0.001, P<0.001, P<0.001, P<0.001respectively) and soft agar colony formation assay (P<0.001, P<0.001, P<0.001, P<0.001respectively). Cells proliferation rates was in a decrease by PHB overexpressed or CLIC4intefered cells to compare with mock cells (P<0.001, P<0.001, P<0.001, P<0.001respectively).(5) The invasive or migrative ability was significantly enhanced in CLIC4and PHB overexpressing cells by comparing with mock cells (P<0.001,P<0.001, P<0.001,P<0.001respectively). And the invasive or migrative ability was significantly weakened in CLIC4and PHB intefered cells, it did not show difference with mock cells (P<0.001, P<0.001, P<0.001, P<0.001respectively).(6) CLIC4overexpressed or PHB intefered cells showed that the tumor growth was significantly in an increase comparing with mock cells as determined by in vivo nude mice subcutaneous tumor forming assay (P<0.05,P<0.05). Pulmonary disseminated foci analysis of mice that received tail vein injections showed that CLIC4overexpressing demonstrated a greater boost in metastatic ability than mock cells. PHB overexpressed cells can not form subcutaneous tumor or pulmonary dissemination.4. The mechanism of colorectal cancer stem cells orchestrating migration through regulating CLIC4and PHB connections(1) The biomarkers of CSC such as CD133, CD44and EpCAM were detected and confirmed by western blot in17CSC with interfered CLIC4or PHB, and in40CSC overexpressed CLIC4or PHB. The results indicated that the expression of CD133, CD44and EpCAM were significantly enhanced in CLIC4or PHB overexpressed comparing with the control cells.(2) Immunoblot results showed that caspase2, caspase3, DFF45, Fas, PARP and RIP were significantly weakened in CLIC4overexpressed and PHB intefered cells comparing with the control cells.(3) CLIC4and PHB overexpressing cells exhibited high expression levels of vimentin, β-catenin, and N-cadherin, and weakly expressed E-cadherin.(4) Confocal laser scanning microscope showed that cytoskeletal proteins F-actin and microtubule were co-stained with CLIC4. F-actin and microtubule were significantly enhanced in CLIC4overexpressing and PHB overexpressing cells comparing with control cells.Conclusion1. PHB interacts with CLIC4, which play roles of migration and metastasis for colorectal CSC.2. PHB interacting with CLIC4also infuences recombination and redistribution of cytoskeletal proteins F-actin and microtubule in CSC. The interaction sites are the67-100amino acids of CLIC4and74-116amino acids of PHB.3. PHB may play a role on ROCK/LIM signaling pathway, inhibition of the degradation of F-actin polymer, maintaining cytoskeletonal stability and promotion of cells’mobility.4. PHB is also involved in the regulation of apoptosis; Upregulated PHB promoted apoptosis and inhibited tumorigenicity of CSC, which demonstrated that the connection of PHB and CLIC4may be influenced by other factors.5. High expression of CLIC4indicates poor prognosis of patients with colorectal cancer. PHB expressed cells resembled to the distribution of CSC in colorectal cancer tissues. PHB was closely associated with tumor location and vessel invasion, suggesting that the interaction of CLIC4and PHB promote CSC to intravasate.New Findings1. Successfully comfirmed the interaction sites of CLIC4and PHB.2. CLIC4is closely correlated with the overall survival of patients. PHB was closely associated with tumor location and vessel invasion.3. CLIC4and PHB connections lead to re-construct cytoskeleton and promote cell migration that may be one of the reasons of metastasis in colorectal CSC.