Design, syntheses and biological evaluation of natural product aiphanol derivatives and analogues: Discovery of potent anticancer agents.

The natural product aiphanol (1) is one of the substances with anticancer biological activity isolated from traditional Chinese medicines (TCM) Smilax glabra Roxb. (Tufuling). Our recent research found that aiphanol could suppress angiogenesis and tumor growth by dual-blocking VEGF/VEGFRs and COX2 signal pathway. In this study, four series of 40 aiphanol derivatives and analogues were designed, synthesized and evaluated for their anticancer activity. Among them, the analogues 10j and 14c exhibited the most potent inhibition and broad-spectrum antiproliferative activity toward nine tumor cell lines. The IC50 values of the analogues 10j and 14c range from 0.81 to 10 µmol/L which up to 80-fold vs. parent compound aiphanol. The structure-activity relationship (SAR) studies indicated that the substrate at 7-position of benzo 1,4-dioxane is very crucial for anticancer activity. Molecular docking indicated that the compound 14c (ent-14c) tightly binds to VEGFR2 and COX2, respectively. Therefore, compounds 10j and 14c could be promising candidates for the development of anticancer agents in the future.

Anti-PAI-1 Monoclonal Antibody Inhibits the Metastasis and Growth of Esophageal Squamous Cell Carcinoma.

Plasminogen activator inhibitor (PAI-1) is highly expressed in esophageal squamous cell carcinoma (ESCC) and strongly contributes to metastasis, making it a potential target for ESCC therapy. However, the antibodies and inhibitors targeting PAI-1 have not shown good therapeutic effect in the in vivo experiments yet. Here, we generated a panel of novel monoclonal antibodies (mAbs) against PAI-1. Analysis of PAI-1 expression in 90 tissue specimens and 128 serum specimens from ESCC patients with these mAbs confirmed that PAI-1 levels was significantly correlated with metastasis and poor survival. In addition, we found that high PAI-1 expression contributed to the enhanced motility and invasiveness of two ESCC cell lines. Next, mAb-1E2 and mAb-2E3, which have highest affinity with PAI-1, were shown to possess strong inhibitory effects on ESCC migration and invasion. Anti-tumor and anti-metastatic effects of mAb-2E3 were further demonstrated in the experimental animal models. Finally, LRP1 was identified as key factor mediating the pro-invasive function of PAI-1 and the anti-invasive capacity of mAb-2E3 in ESCC cells. The mAb-2E3 markedly decreased STAT1 phosphorylation levels and blocked the binding between PAI-1 and LRP1-ClusterII domain. Collectively, mAb-2E3 developed by our lab may be an effective antibody drug which can be used for anti-metastatic therapy in ESCC.

Aiphanol, a multi-targeting stilbenolignan, potently suppresses mouse lymphangiogenesis and lymphatic metastasis.

The high incidence of lymphatic metastasis is closely related to poor prognosis and mortality in cancers. Potent inhibitors to prevent pathological lymphangiogenesis and lymphatic spread are urgently needed. The VEGF-C-VEGFR3 pathway plays a vital role in driving lymphangiogenesis and lymph node metastasis. In addition, COX2 in tumor cells and tumor-associated macrophages (TAMs) facilitates lymphangiogenesis. We recently reported that aiphanol, a natural stilbenolignan, attenuates tumor angiogenesis by repressing VEGFR2 and COX2. In this study, we evaluated the antilymphangiogenic and antimetastatic potency of aiphanol using in vitro, ex vivo and in vivo systems. We first demonstrated that aiphanol directly bound to VEGFR3 and blocked its kinase activity with an half-maximal inhibitory concentration (IC50) value of 0.29 µM in an in vitro ADP-GloTM kinase assay. Furthermore, we showed that aiphanol (7.5-30 µM) dose-dependently counteracted VEGF-C-induced proliferation, migration and tubular formation of lymphatic endothelial cells (LECs), which was further verified in vivo. VEGFR3 knockdown markedly mitigated the inhibitory potency of aiphanol on lymphangiogenesis. In 4T1-luc breast tumor-bearing mice, oral administration of aiphanol (5 and 30 mg· kg-1 ·d-1) dose-dependently decreased lymphatic metastasis and prolonged survival time, which was associated with impaired lymphangiogenesis, angiogenesis and, interestingly, macrophage infiltration. In addition, we found that aiphanol decreased the COX2-dependent secretion of PGE2 and VEGF-C from tumor cells and macrophages. These results demonstrate that aiphanol is an appealing agent for preventing lymphangiogenesis and lymphatic dissemination by synergistically targeting VEGFR3 and inhibiting the COX2-PGE2-VEGF-C signaling axis.

A multi-targeting natural product, aiphanol, inhibits tumor growth and metastasis.

Cancer is one of the main causes of death in humans worldwide, the development of more effective anticancer drugs that can inhibit the malignant progression of cancer cells is of great significance. Aiphanol is a natural product identified from the seeds of Arecaceae and the rhizome of Smilax glabra Roxb. Our preliminary studies revealed that it had potential antiangiogenic and antilymphangiogenic activity by directly targeting VEGFR2/3 and COX2 in endothelial cells. However, the influence of aiphanol on cancer cells per se remains largely undefined. In this study, the effects and related mechanisms of aiphanol on cancer growth and metastasis were evaluated in vitro and in vivo. Acute toxicity assay and pharmacokinetic analysis were utilized to investigate the safety profile and metabolism characteristics of aiphanol. We revealed that aiphanol inhibited the proliferation of various types of cancer cells and the growth of xenograft tumors in mice and zebrafish models. The possible mechanism was associated with the inactivation of multiple kinases, including FAK, AKT and ERK, and the upregulation of BAX and cleaved caspase-3 to promote cancer cell apoptosis. Aiphanol significantly inhibited cancer cell migration and invasion, which was related to the inhibition of epithelial-mesenchymal transition (EMT) and F-actin aggregation. Aiphanol effectively attenuated the metastasis of several types of cancer cells in vivo. In addition, aiphanol exerted no significant toxicity and had fast metabolism. Collectively, we demonstrated the anticancer effects of aiphanol and suggested that aiphanol has potential as a safe and effective therapeutic agent to treat cancer.

Targeting Claudin 18.2 Using a Highly Specific Antibody Enables Cancer Diagnosis and Guided Surgery.

Claudin 18.2 (CLDN18.2) is a new potential target for cancer therapy, especially for advanced gastric cancer (AGC). A molecular targeting probe is of importance for patient stratification and therapeutic guidance. Here, we explored an antibody-dependent molecular imaging strategy for specific detection and surgery guidance based on a CLDN18.2-specific antibody, 5C9. Two imaging probes, 124I-5C9 and Cy5.5-5C9, were synthesized. The specificity to CLDN18.2 being evidenced in the cellular experiments with control, the diagnostic utility was assessed by immunopositron emission tomography (immuno-PET) and fluorescence imaging using xenograft models. A near-infrared fluorescent II imaging probe FD1080-5C9 was designed to facilitate the comprehensive surgical removal of lesions. 124I-5C9 immuno-PET imaging clearly delineated subcutaneous CLDN18.2-positive tumors, with a peak uptake (maximum standardized uptake value; SUVmax) of 2.25 ± 0.30, whereas the highest values for the 124I-IgG and blocking groups were 0.70 ± 0.13 and 0.66 ± 0.12, respectively. Cy5.5-5C9 fluorescence imaging showed similar results. As proof of the diagnosis and guided surgery (DGS) concept, 124I-5C9 and FD1080-5C9 were simultaneously administered in orthotopic CLDN18.2-positive tumor models, facilitating the comprehensive resection of tumor tissue. Combined, 124I-5C9 and FD1080-5C9 are both promising DGS tools: the former reveals CLDN18.2 in lesions as a PET probe, and the latter can guide surgery. These results provide a utility molecular imaging strategy for specific detection and surgery guidance based on a CLDN18.2-specific antibody both in AGC and other cancers.

The biologically functional identification of a novel TIM3-binding peptide P26 in vitro and in vivo.

PURPOSE: Recent studies have shown that TIM3 plays an important role in T-cell failure, which is closely related to the resistance to anti-programmed cell death protein 1 (PD-1) treatment. However, there have been no reports on the application of peptide blockers to TIM3. In this study, we endeavored to identify the in vitro and in vivo anti-tumor activities of a TIM3-targeting peptide screened from the phage peptide library. METHODS: Phage display peptide library technology, surface plasmon resonance, flow cytometry, and mixed lymphocyte reaction were utilized to screen and demonstrate the bioactivities of P26, a TIM3-targeting peptide. Meanwhile, tumor growth assay was performed to evaluate the anti-tumor effect of P26. RESULTS: In terms of affinity, we demonstrated that P26 specifically binds to TIM3 at the cellular and molecular levels, which therefore blocks the interaction between TIM3 and Galectin-9 (Gal-9) and competes with Gal-9 to bind TIM3. Additionally, P26 significantly increases T-cell activity and elevates IFN-γ and IL-2 levels in a dose-dependent manner. Notably, P26 also counteracts Gal-9-mediated T-cell suppression. More importantly, P26 can inhibit growth of MC38-hPD-L1 tumor in mice. CONCLUSIONS: P26, as a novel TIM3-binding peptide, has the ideal bioactivity connecting to TIM3 and the potential prospect of application in immunotherapy as an alternative or adjuvant to existing agents.

Evaluation of 111In-DOTA-F56 peptide targeting VEGFR1 for potential non-invasive gastric cancer xenografted tumor mice Micro-SPECT imaging.

Non-invasive imaging of vascular endothelial growth factor receptor 1 (VEGFR1) remains a great challenge in the early diagnosis of tumors, especially in gastric cancer. Here, we designed and evaluated a novel 111In-DOTA-F56 peptide as a radioactive analogue of F56 (peptide WHSDMEWWYLLG) to bind VEGFR1. It was obtained by radiolabeling DOTA-F56 with 111InCl3 with 98% radiochemical purity and 1.4 ± 0.4 GBq/µmol specific activity. 111In-DOTA-F56 was obtained by the reaction of DOTA-F56 (10 µg) with 111InCl3 in pH 4.0 sodium acetate buffer at 85 °C for 20 min. 111In-DOTA-F56 shows good stability in 0.01 M Phosphate Buffered Saline (PBS) and 5% Human Serum Albumin (HSA). 111In-DOTA-F56 has a high binding affinity for human gastric cancer BGC-823 cells. Bio-distribution studies of 111In-DOTA-F56 were performed in nude mice xenografted with human gastric cancer BGC-823 cells and the results revealed tumor uptake accumulation. A blocking dose of DOTA-F56 significantly reduced the tumor uptake of 111In-DOTA-F56. Tumors were observed with Micro-SPECT images, and the uptake in the tumor increased with time from 4 h to 24 h. The MIP of the Micro-SPECT also showed that the excess DOTA-F56 can specifically block 111In-DOTA-F56 in a mouse tumor model. We successfully synthesized the 111In-DOTA-F56 VEGFR1-targeted peptide as a non-invasive molecule with fine radiochemical properties. Micro-SPECT indicates tumor uptake, which can be further blocked by excess of the F56 peptide, indicating that 111In-DOTA-F56 peptide has potential for early detection of VEGFR1 positive gastric cancer and is worthy of further clinical investigations.

Topoisomerase inhibitors promote cancer cell motility via ROS-mediated activation of JAK2-STAT1-CXCL1 pathway.

BACKGROUND: Topoisomerase inhibitors (TI) can inhibit cell proliferation by preventing DNA replication, stimulating DNA damage and inducing cell cycle arrest. Although these agents have been commonly used in the chemotherapy for the anti-proliferative effect, their impacts on the metastasis of cancer cells remain obscure. METHODS: We used the transwell chamber assay to test effects of Topoisomerase inhibitors Etoposide (VP-16), Adriamycin (ADM) and Irinotecan (CPT-11) on the migration and invasion of cancer cells. Conditioned medium (CM) from TI-treated cells was subjected to Mass spectrometry screening. Gene silencing, neutralizing antibody, and specific chemical inhibitors were used to validate the roles of signaling molecules. RESULTS: Our studies disclosed that TI could promote the migration and invasion of a subset of cancer cells, which were dependent on chemokine (C-X-C motif) ligand 1 (CXCL1). Further studies disclosed that TI enhanced phosphorylation of Janus kinase 2 (JAK2) and Signal transducers and activators of transcription 1 (STAT1). Silencing or chemical inhibition of JAK2 or STAT1 abrogated TI-induced CXCL1 expression and cell motility. Moreover, TI increased cellular levels of reactive oxygen species (ROS) and promoted oxidation of Protein Tyrosine Phosphatase 1B (PTP1B), while reduced glutathione (GSH) reversed TI-induced JAK2-STAT1 activation, CXCL1 expression, and cell motility. CONCLUSIONS: Our study demonstrates that TI can promote the expression and secretion of CXCL1 by elevating ROS, inactivating PTP1B, and activating JAK2-STAT1 signaling pathway, thereby promoting the motility of cancer cells.

Mycoplasma hyorhinis infection promotes gastric cancer cell motility via ß-catenin signaling.

BACKGROUND: We previously identified that Mycoplasma hyorhinis infection promotes gastric cancer cell motility. The ß-catenin signaling pathway is critical to determining malignant cancer cell phenotypes; however, the association between M hyorhinis and the ß-catenin signaling pathway is unclear. METHODS: We performed subcellular fractionation and immunofluorescence staining to observe ß-catenin accumulation in the nucleus. The expression of downstream ß-catenin genes was detected by quantitative RT-PCR. Gastric cancer cell motility was examined by transwell chamber migration and wound healing assays, and a co-immunoprecipitation assay was used to detect the proteins associated with the membrane protein p37 of M hyorhinis. RESULTS: We found that M hyorhinis infection promoted nuclear ß-catenin accumulation and enhanced the expression of downstream ß-catenin genes. M hyorhinis-promoted gastric cancer cell motility was counteracted by treatment with the ß-catenin inhibitor XAV939 or ß-catenin knockdown. We further detected a protein complex containing LRP6, GSK3ß, and p37 in M hyorhinis-infected cells. M hyorhinis also induced LRP6 phosphorylation in a GSK3ß-dependent fashion. Knockdown of LRP6 or GSK3ß abolished M hyorhinis-induced cell motility. CONCLUSION: Our results reveal that the ß-catenin signaling pathway could be activated by M hyorhinis infection, thereby contributing to M hyorhinis-induced gastric cancer cell motility.

PRL-3 Promotes Ubiquitination and Degradation of AURKA and Colorectal Cancer Progression via Dephosphorylation of FZR1.

The oncogenic phosphatase PRL-3 is highly expressed in metastatic colorectal cancer but not in nonmetastatic colorectal cancer or noncolorectal cancer metastatic cancers. Although the proinvasive capacity of PRL-3 has been validated in multiple types of cancer, its impact on colorectal cancer progression and the underlying mechanisms remain poorly understood. Here, we report that overexpressed PRL-3 stimulates G2-M arrest, chromosomal instability (CIN), self-renewal, and growth of colorectal cancer cells in xenograft models, while colorectal cancer cell proliferation is decreased. PRL-3-induced G2-M arrest was associated with decreased expression of Aurora kinase A (AURKA). PRL-3-promoted slow proliferation, CIN, self-renewal, and growth in xenografts were counteracted by ectopic expression of AURKA. Conversely, knockdown of PRL-3 resulted in low proliferation, S-phase arrest, impaired self-renewal, increased apoptosis, and diminished xenograft growth independently of AURKA. Analysis of colorectal cancer specimens showed that expression of PRL-3 was associated with high status of CIN and poor prognosis, which were antagonized by expression of AURKA. PRL-3 enhanced AURKA ubiquitination and degradation in a phosphatase-dependent fashion. PRL-3 interacted with AURKA and FZR1, a regulatory component of the APC/CFZR1 complex. Destabilization of AURKA by PRL-3 required PRL-3-mediated dephosphorylation of FZR1 and assembly of the APC/CFZR1 complex. Our study suggests that PRL-3-regulated colorectal cancer progression is collectively determined by distinct malignant phenotypes and further reveals PRL-3 as an essential regulator of APC/CFZR1 in controlling the stability of AURKA. SIGNIFICANCE: Dephosphorylation of FZR1 by PRL-3 facilitates the activity of APC/CFZR1 by destabilizing AURKA, thus influencing aggressive characteristics and overall progression of colorectal cancer.

Knockdown of PRL-3 increases mitochondrial superoxide anion production through transcriptional regulation of RAP1.

BACKGROUND: Phosphatase of regenerating liver-3 (PRL-3) has been shown to be highly expressed in various types of cancers and is related to poor prognosis. Our previous study showed that silencing of PRL-3 leads to increased reactive oxygen species (ROS). However, the mechanism of PRL-3 regulating ROS is not clear. MATERIALS AND METHODS: PRL-3 or Repressor activator protein 1 (RAP1) was knockdown in human colorectal cancer cell lines HCT116 and SW480. The mRNA level was measured by quantitative real-time (qRT)-PCR and the protein level was measured by western blot. ROS was detected by specific oxidationsensitive fluorescent probes. Cell cycle was analyzed through flow cytometry. Luciferase assay and chromatin immunoprecipitation (ChIP) were performed to investigate the regulation of RAP1 by PRL-3. Gene expression correlation was analyzed through an interactive web server. Statistical analysis was performed with SPSS software. RESULTS: Knockdown of PRL-3 significantly increases mitochondrial superoxide anion, mitochondria membrane potential, and induces cell cycle arrest. Decreased PRL-3-induced mitochondrial superoxide anion accumulation is related to the downregulation of RAP1, which could also affect the level of mitochondria superoxide anion. PRL-3 regulates the expression of RAP1 through binding to the promoter of rap1 gene. PRL-3 could regulate the expression of peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) through the mediation of RAP1. Both PRL-3 and RAP1 could regulate the expression of manganese superoxide dismutase 2 (SOD2) and the uncoupling protein 2 (UCP2), which may be related to PRL-3 suppression induced mitochondria superoxide anion. CONCLUSION: Our study presents the first evidence that PRL-3 is involved in the regulation of mitochondria superoxide anion as a transcriptional factor.

Extracellular gamma-synuclein promotes tumor cell motility by activating ß1 integrin-focal adhesion kinase signaling pathway and increasing matrix metalloproteinase-24, -2 protein secretion.

BACKGROUND: Increasing evidence reveals a significant correlation between gamma-synuclein (SNCG) level and tumor invasion and metastasis in various human cancers. Our previous investigation showed that SNCG could secrete into extracellular environment and promoted tumor cell motility, but the mechanism is unknown. METHODS: The membrane binding ability of SNCG was characterized by immunohistochemical staining, immunofluorescence staining and fractionation of colorectal cancer (CRC) cell membrane. Association between SNCG and ß1 integrin was validated by coimmunoprecipitation and far Western blot. After inhibition of ß1 integrin and focal adhesion kinase (FAK), effect of SNCG on cell motility was measured by transwell chamber assays and changes of protein levels were detected by Western blot. Association between SNCG and activated ß1 integrin levels in human CRC tissues was determined by Spearman's rank correlation analysis. Secreted proteins in conditioned medium (CM) were screened by antibody array. RESULTS: Extracellular SNCG bound ß1 integrin on CRC cell membrane and increased levels of activated ß1 integrin and FAK. Correspondingly, SNCG-enhanced cell motility was counteracted by knockdown or inhibition of ß1 integrin or FAK. Further study revealed that high SNCG level indicated poor outcome and SNCG levels positively correlated with those of activated ß1 integrin and phospho-FAK (Tyr397) in human CRC tissues. Additionally, extracellular SNCG promoted secretion of fibronectin (FN), vitronectin (VN), matrix metalloproteinase (MMP)-2, and MMP-24 from HCT116 cells. Protease activity of MMP-2 in the CM of HCT116 cells was increased by treatment with SNCG, which was abolished by inhibiting ß1 integrin. CONCLUSION: Our results highlight the potential role of SNCG in remodeling extracellular microenvironment and inducing ß1 integrin-FAK signal pathway of CRC cells.

IGHG1 promotes motility likely through epithelial-mesenchymal transition in ovarian cancer.

OBJECTIVE: Ovarian cancer (OC) is one of the leading causes of death for female cancer patients. COC166-9 is an OC-specific monoclonal antibody and we have identified immunoglobulin γ-1 heavy chain constant region (IGHG1) as its antigen. We explore the function of IGHG1 in proliferation, apoptosis and motility of OC cells further in this research. METHODS: IGHG1 expression in OC specimens was detected through immunohistochemistry. Real-time quantitative polymerase chain reaction (RT-qPCR) or western blotting assay was used to test IGHG1 expression in OC cells. Viability of OC cells was tested by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Flow cytometry or western blotting assay was used to detect cell cycle and apoptosis. Cellular motility was analyzed by using transwell assay and the markers of epithelial-mesenchymal transition (EMT) were tested through immunoblots. RESULTS: Although it exerts negligible effect on the viability and apoptosis of OC cells, IGHG1 could promote migration and invasion of malignant cells in vitro. Mechanistically, IGHG1 increases the expression of N-cadherin and Vimentin while decreases E-cadherin expression. Additionally, IGHG1 expression in OC specimens is higher relative to the paired normal counterparts. Further analysis demonstrates that the increased IGHG1 expression correlates positively with the lymph node metastasis of OC. CONCLUSIONS: IGHG1 promotes the motility of OC cells likely through executing the EMT program. Increased IGHG1 expression in OC specimens is associated with the lymph node metastasis.

Development of a novel albumin-based and maleimidopropionic acid-conjugated peptide with prolonged half-life and increased in vivo anti-tumor efficacy.

Angiogenesis plays a critical role in tumor aggressiveness, and a lot of anti-angiogenic agents have been used in clinical therapy. The therapeutic efficacy of peptides are generally restricted by the short in vivo life-time, thus, we were interested in developing a novel albumin-based and maleimidopropionic acid-conjugated peptide to prolong the half-life and improve the anti-tumor effect. Methods: We developed a peptide F56 with a maleimidopropionic acid (MPA) at the C-terminal (denoted as F56-CM), which allows immediate and irreversible conjugation with serum albumin. Biological property and anti-tumor activity of F56-CM were evaluated in vitro and in vivo. Results: We showed that F56-CM reduced migration and tube formation of endothelial cells in vitro and inhibited the generation of subintestinal vessels (SIV) in zebrafish embryos in vivo. F56-CM inhibited vascular endothelial growth factor (VEGF) induced phosphorylation of VEGFR1 and activation of the PI3K-AKT axis. Furthermore, F56-CM rapidly conjugated with albumin upon intravenous injection and extended the biological half-life of F56 from 0.4249 h to 6.967 h in rats. Compared with F56, F56-CM exhibited stronger anti-tumor activity on both BGC-823 gastric cancer and HT-29 colon cancer xenografts in nude mice, and the statistical difference was remarkable. More significantly, the efficacy of F56-CM inhibiting lung metastasis of BGC-823 cells was also better than that of F56. The inhibition rates were 62.1% and 78.9% for F56 and F56-CM respectively when administrated every day, and 43.8% and 63.1% when administrated every four days at equal dose. Conclusions: Taken together, our results demonstrated that F56-CM has considerable potential for cancer therapy.

Sarsaparilla (Smilax Glabra Rhizome) Extract Activates Redox-Dependent ATM/ATR Pathway to Inhibit Cancer Cell Growth by S Phase Arrest, Apoptosis, and Autophagy.

Sarsaparilla (Smilax Glabra Rhizome) exerts growth inhibitory effect on multiple cancer cells in vitro and in vivo, and redox-dependent persistent activation of ERK1/2 has been reported to underlie this effect. Here, we report an activation of ATM/ATR-dependent signaling pathway also as a mechanism for the cancer cell growth inhibition induced by the supernatant fraction of the water-soluble extract from sarsaparilla (SW). SW treatment (3.5 µg/µL) promoted the phosphorylations of ATM, ATR, and CHK1 in AGS and HT-29 cells. The ATM kinase inhibitor, KU55933, could reverse SW-induced ERK phosphorylation but not the reduced glutathione/oxidized glutathione (GSH/GSSG) imbalance in AGS cells. However, both the redox inhibitor glutathione (GSH) and ERK inhibitor U0126 antagonized SW-induced phosphorylations of ATM, ATR, and CHK1 in AGS cells. We further found KU55933 significantly antagonized SW-induced S phase arrest, apoptosis, autophagy and the resultant cell growth inhibition. Our results provide another molecular basis for the anticancer action of sarsaparilla.

PRL-3 promotes telomere deprotection and chromosomal instability.

Phosphatase of regenerating liver (PRL-3) promotes cell invasiveness, but its role in genomic integrity remains unknown. We report here that shelterin component RAP1 mediates association between PRL-3 and TRF2. In addition, TRF2 and RAP1 assist recruitment of PRL-3 to telomeric DNA. Silencing of PRL-3 in colon cancer cells does not affect telomere integrity or chromosomal stability, but induces reactive oxygen species-dependent DNA damage response and senescence. However, overexpression of PRL-3 in colon cancer cells and primary fibroblasts promotes structural abnormalities of telomeres, telomere deprotection, DNA damage response, chromosomal instability and senescence. Furthermore, PRL-3 dissociates RAP1 and TRF2 from telomeric DNA in vitro and in cells. PRL-3-promoted telomere deprotection, DNA damage response and senescence are counteracted by disruption of PRL-3-RAP1 complex or expression of ectopic TRF2. Examination of clinical samples showed that PRL-3 status positively correlates with telomere deprotection and senescence. PRL-3 transgenic mice exhibit hallmarks of telomere deprotection and senescence and are susceptible to dextran sodium sulfate-induced colon malignancy. Our results uncover a novel role of PRL-3 in tumor development through its adverse impact on telomere homeostasis.

Repressor activator protein 1-promoted colorectal cell migration is associated with the regulation of Vimentin.

Repressor activator protein 1 plays important roles in telomere protection, while repressor activator protein 1 binds to extra-telomeric DNA and exerts the function as a transcriptional regulator. Previous study showed that repressor activator protein 1 regulates the transcriptional activity of nuclear factor-κB, and it was highly expressed in breast cancer tissues; however, the clinical significance of repressor activator protein 1 expression in cancer remains to be elucidated. In this study, we discovered that repressor activator protein 1 was highly expressed in colorectal cancer tissues. High expression of repressor activator protein 1 was significantly correlated with poor prognosis and distant metastasis. Knockdown of repressor activator protein 1 in colorectal cancer cells did not affect cell proliferation or colony formation, but dramatically decreased cell migration and F-actin-enriched membrane protrusions. Microarray screening revealed that Vimentin was downregulated after repressor activator protein 1 knockdown, which was validated by analysis of a colorectal cancer dataset. Furthermore, knockdown of Vimentin attenuated repressor activator protein 1-enhanced cell migration. Thus, our study suggests that repressor activator protein 1 is a prognostic marker and a potential target for colorectal cancer therapy.

Gene regulatory pattern analysis reveals essential role of core transcriptional factors' activation in triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype. Genome-scale molecular characteristics and regulatory mechanisms that distinguish TNBC from other subtypes remain incompletely characterized. RESULTS: By combining gene expression analysis and PANDA network, we defined three different TF regulatory patterns. A core TNBC-Specific TF Activation Driven Pattern (TNBCac) was specifically identified in TNBC by computational analysis. The essentialness of core TFs (ZEB1, MZF1, SOX10) in TNBC was highlighted and validated by cell proliferation analysis. Furthermore, 13 out of 35 co-targeted genes were also validated to be targeted by ZEB1, MZF1 and SOX10 in TNBC cell lines by real-time quantitative PCR. In three breast cancer cohorts, non-TNBC patients could be stratified into two subgroups by the 35 co-targeted genes along with 5 TFs, and the subgroup that more resembled TNBC had a worse prognosis. METHODS: We constructed gene regulatory networks in breast cancer by Passing Attributes between Networks for Data Assimilation (PANDA). Co-regulatory modules were specifically identified in TNBC by computational analysis, while the essentialness of core translational factors (TF) in TNBC was highlighted and validated by in vitro experiments. Prognostic effects of different factors were measured by Log-rank test and displayed by Kaplan-Meier plots. CONCLUSIONS: We identified a core co-regulatory module specifically existing in TNBC, which enabled subtype re-classification and provided a biologically feasible view of breast cancer.