The Relationship Between TIP30 And Tumorigenesis And Tumor Prognosis

Background and objectives:Lung cancer is one of the most harmful diseases around the world today and ranks as the first cause of cancer-related deaths. Several proto-oncogenes and tumor suppressor genes, such as EGFR, k-Ras, c-Myc, p53 and DOK have been implicated in the pathogenesis of non-small cell lung cancer (NSCLC). The emergence of EGFR kinase inhibitor, monoclonal antibody and novel chemotherapeutic drugs, developments in radiotherapy technology and improvement of surgical methods have made great progress in the treatment of lung malignancy, but the overall prognosis of patients remains poor, among which the 5-year survival rate of those with NSCLC is only around 16%, and the outcomes of patients with small cell lung cancer(SCLC) is even worse. For decades, the mortality rate of lung carcinoma is keeping high, mainly due to lack of effective methods for early diagnosis and treatment which can significantly prolong the survival of advanced-stage patient. According to statistics, 85% of lung cancer is NSCLC which consists of 40% adenocarcinoma.50% patients with lung cancer overexpressed epidermal growth factor receptor (EGFR), an important molecule which can mediate cellular growth signal through activation of downstream signaling molecules induced by kinase phosphorylation and effecting on functional gene after nuclear internalization. Significantly correlating with the carcinogenesis, development and prognosis of lung cancer, EGFR has become the most crucial target for lung cancer therapy. Various EGFR targeted therapeutic drugs including gefitinib, erlotinib and cetuximab have been approved for clinical use in the treatment of lung cancer. Gefitinib/erlotinib shows high effective rates in patients bearing EGFR mutation in exon 19 or 21 but relatively low efficiencies in patients carrying no mutation in exon 19 or 21. However, only a minority of patients with lung adenocarcinoma carry EGFR mutation, most of which could not get a satisfactory remission, urging us to find new therapeutic targets and methods for the improvement of effective treatment of lung cancer. Thus, it is of great importance to further explore the regulatory mechanism of EGFR signaling for detection of new mechanisms which would help to find new therapeutic targets and enhance the effective treatments.Primary hepatic carcinoma ranks the fifth cause of cancer-related deaths, over 50% of which arises in China and seriously damage the health of Chinese people. The incidence of most primary hepatic carcinoma is due to chronic hepatic injury and repair leading by hepatitis virus infection, chronic alcoholic hepatitis and other toxicants, following hepatofibrosis and induction of hepatoma. It has been reported that damage-induced hepatic inflammatory repair plays a key role in the occurrence of hepatoma, just as NFκB/IL-6/STAT3 signaling performs essential functions in the repair of injury and tumorgenesis of hepatoma. The central cause for hepatoma being the most lethal malignancies is the difficulties in early diagnosis and lack of effective treatments for advanced stage patients. Recently, molecular targeted therapy has achieve certain success in the treatment of hepatoma, but patient benefits limited survival rates, thus further studies on the mechanism is needed to discover novel pathogenesis and therapeutic targets of hepatoma.TIP30, also named HTATIP2 (HIV-1 Tat interactive protein 2), is a 30KD Tat (Transactivator of transcription) interactive protein and first found by Xiao in the study of in vitro transcription of HIV (human immunodeficiency virus). As a cofactor for the transcription of HIV, TIP30 could specifically promote the proliferation of HIV by interacting with Tat. Later sequence analysis showed that TIP30 is the same protein reported as the expression of anti-metastasis gene CC3 in the same period. Evidence suggested that TIP30 could inhibit the. metastasis of tumor through regulating relevant genes for cellular proliferation, apoptosis and angiogenesis. But latest researches found that, besides for regulating gene transcription, TIP30 performs multiple functions as regulate DNA damage and repair, impact cellular glucose tolerance and adjust molecular transport within the cytoplasm. The study of TIP30 has entered a new period of rapid development; related works are prone to obtain breakthroughs and facilitate our investigation of new mechanism underlying the carcinogenesis of lung cancer. Whether or not TIP30 could be established as a novel targets for the treatment of malignancies merits intense attention.Existing researches have proved that TIP30 is significantly correlated with the metastasis of lung cancer and can suppress in vitro growth of lung cancer cells. In the context of mixed mice strains, knockout of Tip30 led to spontaneous occurrence of hepatoma. Together with the fact that 37% of human primary hepatic cancer is associated with downregulation of TIP30, suggesting TIP30 as a crucial part in the development of hepatoma. At present, direct evidence is little about the relation between TIP30 and the occurrence as well as prognosis of lung cancer and hepatoma, let alone reports involving relevant mechanisms. Among the questions that remain to be resolved are that whether knockout of Tip30 can still induce spontaneous hepatoma and the relation between Tip30 and lung cancer in homozygous background.Our study aimed at investigating the relation and relevant mechanisms concerning knockout of Tip30 and development of lung cancer and hepatoma, analyzing clinical data from current available microarray database and exploring the correlation between TIP30 and prognosis as well as susceptibility of lung cancer, thus confirm the role of TIP30 in tumorgenesis and metastasis.Methods:1. Animal breeding and processingObtained Tip30 knocking-out Balb/c or FBV mice strain by crossbreeding C57J6 mice knocking-out Tip30 with Balb/c or FBV mice, finally established Tip30 knocking-out mice with either Balb/c or FBV after over 7 generations continuous passage. After being observed for 18 months of disease, death and tumorgenesis, mice were sacrificed for relevant research.2. HistologyThe lungs, liver, spleen and mammary gland of mice were dissected and routinely fixed in 10% formalin, following rehydrated in graded alcohols and paraffin embed. After final tissue sectioning and H & E staining, slides were examined by two pathologists for tumorgenesis or other aberrant histological changes.3. ImmunohistochemistryImmunohistochemistry was performed as described by the protocol supplied by Cell Signaling Technology.3-4μm lung sections from the embedded blocks were deparaffinized with dimethyl benzene, rehydrated in graded alcohols, and subject to heat-induced epitope retrieval in dimethyl benzene in a microwave. After treating with H2O2 for neutralizing hydrogen peroxidase and blocking with 5% BSA, the slides were incubated with a primary antibody over night at 4℃. After being incubated with biotin conjugated second antibodies, sections were treated with ABC reagent, stained with DAB, counterstained the nuclei with hematoxylin and mounted with permanent blocking solution for microscopic observation.4. Immunofluorescence/confocal scanning microscopyTissue immunofluorescence:Immunofluorescent analysis was performed as immunochemistry. After being sliced, deparaffinized, rehydrated, epitope retrieval and blocked, sections were incubated with primary antibody overnight and secondary antibody with fluorescein label for 1h, following mounting in 0.2% sudan black to reduce autofluorescence of the tissue. Chromatin was counterstained with DAPI (4'6-diamidino-2-phenylindole) and slides coverslipped before observing under a fluorescence microscope.In vitro confocal laser scanning microscopy:Cell climbing sheet were dealt in different time, fixed in 4% paraformaldehyde, dipped in 0.1% Triton-100, blocked with 5% BSA, incubated with primary antibodies overnight and secondary antibody with fluorescein labeling for 1h. After counterstained chromatin with DAPI, sections were coverslipped and observed under confocal laser scanning microscope.5. Western blotTissue/cellular protein extraction:Prepared tissues were diced into small pieces (cells agita bene) and lysed in RIPA buffer (containing 1mMDTT and protease inhibitor) at 4℃for 20min. Lysates were centrifuged at 13000RPM for 10min. Determine the concentration of the supernatant before mixing with an equal volume of electrophoresis sample buffer and then boiled for supernatant.Nuclear/cytoplasmic protein extraction:Performed according to the manufacturer's protocol (Millipore).Detection:Mixed samples electrophoresed by SDS-PAGE, transferred to PVDF membranes, blocked with 5% BSA and incubated with primary antibodies at 4℃overnight. Interacted secondary antibodies for 45min, washed before being scanned by odyssey imaging system and analyzed by Odyssey 2.1 software.6. Gene transfectionLentiviruses were generated using available plasmids and then transfected cells following puromycin selection. Transfection efficiencies were analyzed by western blot.7. Cell apoptosis assayApoptotic cells were visualized using a TUNEL assay kit (Roche) according to the manufacture's instruction. Numbers of TUNEL-positive cells in the lung were counted from five randomly selected high-power fields per sample.8. Cell growth assayCells were seeded and cultured in 96-well plates. Cell viability was measured according to different groups and times. After being incubated with 10μl CCK-8 for 1h at 37℃, the cells were was measured for OD values (584nm).9. Fluorescent quantitative PCR (polymerase chain reaction)Total RNAs of liver tissues were isolated using RNA Extraction Kit from Qiagen Company and cDNAs were reversely transcripted from mRNA using oligodT, dNTP and ReversetranscriptaseⅢfrom Invitrogen Company. QPCR were performed using Sybr Green qPCR Kit from Biorad and RNA polymeraseⅡas tissue control. The primer sequences used were as follows:SP-1,5'-TCCATGGATGAAGTGACAGC TG-3',5'-TGGGAGTTGTTGCTGTTCTCAT-3'; IL-6,5'-AGAGGAGACTTC ACAGAGGAT-3',5'-TACTCCAGGTAGCTATGGTAC-3'. PCR amplification was performed using Biorad iQ5 after an initial denaturation of 3 min at 95℃, followed by 40 cycles of 30s at 95℃,30s at 55℃and 30s at 72℃, and final elongation at 72℃for 30s. The relative mRNA levels between experimental and control samples (R) were determined using the formula R=2-(Ct(experimental sample)-Ct (control sample).10. Microarray assay analysisAnalyzed an open published microarray dataset involving 442 human lung adenocarcinoma which is for now the largest study. Final value of TIP30 was average of 3 groups. Medians of TIP30 were calculated, patients with exceeding TIP30 were accounted as TIP30 high expression group and those with lower TIP30 low expression. Survival times, relapse times and death times before relapse were calculated and survival curves were made to compare differences between these two groups.11. Statistical analysis.Results were indicated as mean±SD (x±s) and statistical analyzed with SPSS 13.0 software. Comparisons of differences in various molecular expressions, incidences and survival curves between two groups of mice were analyzed with independent t test, x2 test and Log-rank (mantel-cox) test respectively.Results:1. Knockout of Tip30 led to the spontaneous occurrence of lung tumor in Balb/c miceIn the observation of 18 months, most of the 51 Balb/c mice with Tip30-/- were associated with proliferation in alveolar epithelium, of which 43.2% developed spontaneous lung tumor (22/51), significantly more than the 8.3%(1/12) of Tip30+/-mice and 4.2% of Tip30+/+ mice.2. Spontaneous formed lung tumor by Tip30 depletion possessed unique charactersHistological observation found that most of spontaneous formed lung tumor led by Tip30 knockout was lung adenoma (10/51) or adenocarcinoma (12/51), locating in the periphery of lung. Immunochemical analysis showed that marker SP-C of type II alveolar epithelial cell stains positive and CC10 of bronchi epithelial cell Clara negative in all spontaneous formed lung tumor, suggesting that tumors origin from type II epithelial cells.3. Tip30 promoter was mainly activated in type II alveolar epithelial cellDouble staining immunofluorescence techniques found thatβ-Gal highly expressed in SP-C positive cells (typeⅡalveolar epithelial cell) but not in CC10 positive ones. Since Tip30 promoter were reserved andβ-Gal was inserted into original Tip30 locus when Tip30 was knocking out, above results suggests that Tip30 may overexpressed in typeⅡalveolar epithelial cell and low in bronchiolar epithelium Clara cells.4. Tip30 knockout bronchioles led to increase of stem cells/progenitor cells in bronchoalveolar duct junctionSP-C and CC10 positive cells in bronchoalveolar duct junction are lung epithelial stem cells/or progenitor cells. Double immunofluorescence staining results showed that SP-C and CC10 double-positive cells in the bronchoalveolar duct junction of the Tip30-/- mice are remarkably more than those of Tip30+/+ mice, P<0.05.5. Spontaneous lung tumor was led by Tip30 knockout through promoting proliferation of type II alveolar epithelial cellImmunohistochemistry examination showed that the number of typeⅡalveolar epithelial cells per high power field in Tip30-/- mice was significantly more than those in Tip30+/+, P<0.05. Double immunofluorescence staining results showed that PCNA positive expression of type II alveolar epithelial cells were significantly more in Tip30-/- mice comparing with those in Tip30+/+ mice, P<0.05. The number of apoptotic cells of alveolar cell in Tip30-/- mice showed no significant difference comparing with those in Tip30+/+ mice, P>0.05.6. Tip30 knockout promoted proliferation of pulmonary epithelial cells through upregulating EGFR and activating the downstream signaling molecules of EGFRImmunofluorescence examination showed that spontaneously formed lung tumor induced by Tip30 knockout all positive express EGFR and the number of EGFR positive cells of alveolar in Tip30-/- mice were significantly increased comparing with Tip30+/+ mice, P<0.05. Immunochemical study proved that expression of pAkt,pErk and cyclin D1 of alveolar and tumor in Tip30-/- mice were significantly enhanced, P<05. Western blot also confirmed that expression of pAkt and pErk in lung tissue of Tip30-/- ice were significantly stronger than Tip30+/ ice, P<0.05.7. TIP30 knockdown could prolong the activation of EGFR downstream signaling molecules of human lung adenocarcinoma inducing by EGFWestern blot showed that the level of pAKT in lung adenocarcinoma cell A549 downregulating TIP30 (TIP30-SH1) is significantly higher than control cell (shRNA-CON) after induction by EGF for 2h, P<0.05. After inducing by EGF for 8h, the level of pERK in TIP30-SH1 cell was significantly elevated as compared to shRNA-CON cell, P<0.05.8. TIP30 knockdown suppressed EGFR degradation by interfered with the transport of EGFR in early endosomesImmunofluorescence/confocal laser scanning microscopy showed that, after EGF induction, EGFR of lung adenocarcinoma cell A549 whose TIP30 is inhibited stagnates in EEA1-positive early endosomes to prevent selection of EGFR for intralysosomal degradation. Western blot results that EGFR level of TIP30-SH1 cell is significantly enhanced comparing to shRNA-CON cell, P<0.05, suggesting that EGFR degradation is notably suppressed in lung adenocarcinoma cell A549 in which TIP30 is inhibited.9. TIP30 knockdown could promote nuclear internalization of EGFR induced by EGFImmunofluorescence/confocal scanning microscopy found that nuclear internalization of EGFR in TIP30-inhibitory A549 significantly enhanced after inducing by EGF for 2h. Western blot showed that nuclear internalization of EGFR in TIP30-SH1 significantly increased comparing with shRNA-CON, P<0.05. Thus, TIP30 may abate EGF-induced nuclear internalization of EGFR.10. EGF can lead to nuclear internalization of TIP30Immunofluorescence/confocal scanning microscopy showed that TIP30 itself exhibits notable nuclear internalization after EGF induction. 11. Early stage lung adenocarcinoma patients with low expression of TIP30 had better prognosisAnalysis of one of the largest published microarray dataset involving 442 lung adenocarcinomas with Kaplan-Meier method showed that the overall survival time of early lung adenocarcinoma patients whose TIP30 expression level fall below median is significantly prolonged than those with high expression of TIP30, P<0.05.12. Lung adenocarcinoma patients with low expression of TIP30 survived longer after progressionAnalysis of patients with recurrent early lung adenocarcinoma after surgery showed that, patients with TIP30 expression below median had significant longer survival times after recurrence as compared with those with elevated TIP30 expression, P<0.05. The median time from recurrence to death of these patients was 19 months, significantly longer than patients with TIP30 expression above median (7.5 months).13. Expression of TIP30 was correlated with sensitivities to EGFR kinase inhibitorsIn vitro inhibition experiments showed that inhibition rate of TIP30-SH1 by EGFR kinase inhibitor gefitinib and MEK/ERK inhibitor U0126 is significantly larger than shRNA-CON cells after 5 days administration, P<0.05. Thus abaissement of TIP30 could confer more sensitivity to human lung adenocarcinoma cell A549 for EGFR kinase inhibitor gefitinib and MEK/ERK inhibitor U0126.14. Tip30 knockout could not induce spontaneous liver tumor in mice with homozygous backgroundWe found that 51 Balb/c homozygous and 32 FBV homozygous mice with Tip30 knockout exhibited no spontaneous liver tumor, differing from previous reports that Tip30 knockout in homozygous mice could cause spontaneous liver tumors.15. Knockout of Tip30 could prevent liver tumor inducing by DENLiver tumor incidence of male FBV mice with Tip30+/+ after peritoneal injection of 5mg/Kg nitrosodiethylamine for 8 months was 90.9%(10/11), whereas 13 male FBV mice with Tip30-/- developed no liver malignancies (0/13). The difference between two experimental groups was significant, P<0.05.16. Knockout of Tip30 could repress DEN-induced hepatic fibrosis of miceImmunochemistry performing after administration of 100mg/kg Den for 6 days or 5mg/kg for 8 months showed thatα-SMA expression in the liver of Tip30-/- mice was significantly decreased as compared with those of Tip30+/+ mice. Hence, knockout of Tip30 could inhibit the hepatic fibrosis inducing by DEN.17. Knockout of Tip30 could suppress the proliferation of liver tissue and cellAfter administration of 100mg/kg DEN for 6 days, Tip30-/- mice showed significantly lower rate of liver/body weight than Tip30+/+ mice, P<0.05. Immunochemistry showed that PCNA-positive cell in liver of Tip30-/- mice is significantly less than Tip30+/+ mice, P<0.05. Western blotting later confirmed the result.18. Knockout of Tip30 had no discernible effect on DEN-induced hepatic apoptosis of mice.After being dealt with 100mg/kg DEN for 4h or 5mg/kg DEN for 8 months, Tip30-/- mice had slightly more apoptotic cells than Tip30+/+ mice, but the difference between two groups was not significant, P>0.05.19. Knockout of Tip30 inhibited expression of cyclin D1 in hepatocytesAfter administration of 100mg/kg DEN for 3 days or 5mg/kg DEN for 8 months, Tip30-/- mice showed significant less cyclin D1-positive cells than Tip30+/+ mice in immunochemistry experiments. Western blotting further confirmed these results.20. Knockout of Tip30 suppressed the activation of STAT3Western blot examination showed that hepatocellular expression of pSTAT3 in Tip30-/- mice was decreased as compared with that in Tip30+/+ mice after administration of DEN and the difference was significant, P<0.05.21. Knockout of Tip30 downregulated DEN-induced transcription and translation of IL-6Fluorescent quantitative PCR proved that IL-6 mRNA expressing in the liver of Tip30-/- mice is significantly weaker than Tip30+/+ mice after dealing with DEN for 4h. ELISA detection found that expression of serum IL-6 protein in Tip30-/- mice is significantly lower than Tip30+/+ mice after administration of DEN for 24h, P<0.05.22. Knockout of Tip30 suppressed the activation of NFκBWestern blot examination showed that hepatocellular expression of pNFκB in Tip30-/- mice was decreased as compared with that in Tip30+/+ mice after administration of DEN and the difference was significant, P<0.05.23. Tip30 knockout suppressed the degradation of IκB induced by the treatment of DENWestern blot examination showed that hepatocellular expression of IκB in Tip30-/- mice was higher than that of Tip30+/+ mice after treatment of DEN and the difference was significant, P<0.05.24. Knockout of Tip30 repressed infiltration of macrophage in the liver led by DEN-induced hepatic damageImmunochemistry analysis showed that the numbers of MAC-2-positive macrophage between Tip30-/- and Tip30+/+ mice is not significantly different following administration of 100mg/kg DEN for 24h. After dealt with 100mg/kg DEN for 72h or 5mg DEN for 8 months, MAC-2-positive macrophage in the liver of Tip30-/- mice is significantly less than that of Tip30+/+ mice and the difference between two groups was not significant, P<0.05.25. Tip30 promoter was mainly activated in macrophagesDouble staining immunofluorescence results indicated thatβ-Gal expresses highly in MAC-2-positive (Kupffer's cells) hepatocytes, which may due to the insert ofβ-Gal in the original Tip30 gene locus when knocking out Tip30 and meanwhile preserving the Tip30 promoter. Therefore, Tip30 may express highly in Kupffer's cells.26. Knockout of Tip30 downregulated DEN-induced transcription and translation of SP-1Fluorescent quantitative PCR suggested that SP-1 mRNA of hepatocyte in Tip30-/- mice expressed significantly low as compared with that in Tip30+/+ mice, P<0.05.27. Knockout of Tip30 imposed no effect on the activity of downstream signaling molecules of EGFR in mice hepatocytes after DEN administrationWestern blotting examination showed that the difference in expression of pAkt and pErk between hepatocyte of Tip30-/- and Tip30+/+ mice is not significant, P>0.05.Conclusion:1. Tip30 is a multifunctional protein which acts in a tissue-specific and cell-specific pattern. Tip30 could inhibit occurrence of lung cancer and promote DEN-induced hepatoma.2. Tip30-knockout mice is a unique animal model of lung cancer, in which all the tumors arises is lung adenoma or lung adenocarcinoma locating in peripheral lung, origining from typeⅡalveolar epithelial cell and associating with expression of EGFR as well as activation of downstream signaling pathways.3. Knockout of Tip30 can stimulate the growth of typeⅡalveolar epithelial cell and lead to spontaneously-formed lung adenocarcinoma through increasing the number of lung epithelial stem cell/progenitor cell, upregulating expression of EGFR and enabling the activation of EGFR downstream signaling molecules.4. Inhibition of Tip30 could interfere with the sorting of EGFR from endosome to lysosome and attenuate EGF-induced degradation of EGFR, thus prolong activating time of EGFR downstream signaling molecules and promote the proliferation of lung cancer cells.5. Inhibition of Tip30 could facilitate the EGF-induced nuclear internalization of EGFR and accelerate expression of cyclin D1, hence tune the growth of lung cancer cells.6. The expression level of Tip30 could be used as a prognostic indicator for early lung adenocarcinoma:downregulation of Tip30 is associated with longer survival time after surgery and recurrence for early lung adenocarcinoma patients.7. Lung adenocarcinoma cells decreased expressing Tip30 is more sensitive to EGFR kinase inhibitor gefitinib and is thus a potent effective clinical predictors for EGFR kinase inhibitor gefitinib. 8. Knockout of Tip30 could cause no spontaneous liver tumor in homozygous mice and prevent induction of spontaneous liver tumors by chemicals DEN.9. Knockout of Tip30 could restrain DEN-induced carcinogenesis of hepatoma through downregulating activities of NFκB/IL-6/STAT3 signaling pathway and containing DEN-induced hepatocellular injury and repair.10. Knockout of Tip30 hinders DEN-induced hepatic fibrosis and carcinogenesis of hepatoma by downregulating SP-1 and reducing DEN-induced hepatic infiltration of macrophage after hepatocellular injury.