Identification of metastasis-associated exoDEPs in colorectal cancer using label-free proteomics

Exosomes are secreted nanovesicles consisting of biochemical molecules, including proteins, RNAs, lipids, and metabolites that play a prominent role in tumor progression. In this study, we performed a label-free proteomic analysis of exosomes from a pair of homologous human colorectal cancer cell line with different metastatic abilities. A total of 115 exoDEPs were identified, with 31 proteins upregulated and 84 proteins downregulated in SW620 exosome. We also detected 30 proteins expressed only in SW620 exosomes and 60 proteins expressed only in SW480 exosomes. Bioinformatics analysis enriched the components and pathways associated with the extracellular matrix, cytoskeleton-related pathways, and immune system changes of colorectal cancer (CRC). Cellular function experiments confirmed the role of SW620 exosomes in promoting the proliferation, migration, and invasion of SW480 cells. Further verifications were performed on six upregulated exoDEPs (FGFBP1, SIPA1, THBS1, TGFBI, COL6A1, and RPL10), three downregulated exoDEPs (SLC2A3, MYO1D, and RBP1), and three exoDEPs (SMOC2, GLG1, and CEMIP) expressed only in SW620 by WB and IHC. This study provides a complete and novel basis for exploring new drug targets to inhibit the invasion and metastasis of CRC.     

The mechanisms underlying the enrichment and action of glypican-1-positive exosomes in colorectal cancer cells

BackgroundGlypican-1 (GPC1) is overexpressed in several tumors, and GPC1⁺ exosomes have shown the potential to predict early colorectal cancer (CRC). However, the mechanisms underlying the enrichment and action of GPC1⁺ exosomes in CRC remain unknown.MethodsThe expression of slit guidance ligand 2 (SLIT2), hypoxia-inducible factor (HIF)-1α/2α, and GPC1 in clinical CRC tissues was detected using immunohistochemistry and western blot. Exosomes were isolated from the supernatants of CRC cell cultures. The effects of SLIT2, hypoxia, heparin, and phospholipase C (PLC) on exosomal GPC1 expression and GPC1⁺ exosome enrichment in CRC cells were analyzed with western blot and flow cytometry. CRC cell proliferation was assessed with MTT and colony formation assays. Co-immunoprecipitation was used to detect the binding of GPC1 and SLIT2 in SW480 cells. Nude mice were subcutaneously inoculated with SW480 cells with different treatments. The Wnt signaling was detected.ResultsSLIT2 was poorly expressed and GPC1, HIF-1α, and HIF-2α were highly expressed in human CRC tissues. SLIT2 in CRC cells inhibited GPC1⁺ exosome enrichment and exosomal GPC1 expression. PLC and heparin increased GPC1⁺ exosome enrichment in CRC cells in a concentration-dependent manner. Hypoxia increased the enrichment of GPC1⁺ exosomes in CRC cells depending on HIF-2α expression. GPC1⁺ exosomes stimulated CRC cell proliferation and xenograft tumor growth through activation of Wnt signaling.ConclusionsGPC1⁺ exosome enrichment is related to PLC and heparin. Hypoxia increases the enrichment of GPC1⁺ exosomes in CRC cells by activating HIF-2α and downregulating SLIT2. GPC1⁺ exosomes further drive CRC progression by activating Wnt signaling.     

Exosomes Derived from Human Primary and Metastatic Colorectal Cancer Cells Contribute to Functional Heterogeneity of Activated Fibroblasts by Reprogramming Their Proteome

Cancer‐associated fibroblasts (CAFs) are a heterogeneous population of activated fibroblasts that constitute a dominant cellular component of the tumor microenvironment (TME) performing distinct functions. Here, the role of tumor‐derived exosomes (Exos) in activating quiescent fibroblasts into distinct functional subtypes is investigated. Proteomic profiling and functional dissection reveal that early‐ (SW480) and late‐stage (SW620) colorectal cancer (CRC) cell‐derived Exos both activated normal quiescent fibroblasts (α‐SMA^?, CAV⁺, FAP⁺, VIM⁺) into CAF‐like fibroblasts (α‐SMA⁺, CAV^?, FAP⁺, VIM⁺). Fibroblasts activated by early‐stage cancer‐exosomes (SW480‐Exos) are highly pro‐proliferative and pro‐angiogenic and display elevated expression of pro‐angiogenic (IL8, RAB10, NDRG1) and pro‐proliferative (SA1008, FFPS) proteins. In contrast, fibroblasts activated by late‐stage cancer‐exosomes (SW620‐Exos) display a striking ability to invade through extracellular matrix through upregulation of pro‐invasive regulators of membrane protrusion (PDLIM1, MYO1B) and matrix‐remodeling proteins (MMP11, EMMPRIN, ADAM10). Conserved features of Exos‐mediated fibroblast activation include enhanced ECM secretion (COL1A1, Tenascin‐C/X), oncogenic transformation, and metabolic reprogramming (downregulation of CAV‐1, upregulation of glycogen metabolism (GAA), amino acid biosynthesis (SHMT2, IDH2) and membrane transporters of glucose (GLUT1), lactate (MCT4), and amino acids (SLC1A5/3A5)). This study highlights the role of primary and metastatic CRC tumor‐derived Exos in generating phenotypically and functionally distinct subsets of CAFs that may facilitate tumor progression.     

Circulating Exosomal miR-17-5p and miR-92a-3p Predict Pathologic Stage and Grade of Colorectal Cancer

Exosomes are extracellular membrane vesicles of 50- to 130-nm diameter secreted by most tumor cells. Exosomes can mediate the intercellular transfer of proteins and RNAs, including microRNAs (miRNAs), and promote both tumorigenesis and premetastatic niche formation. In this study, we performed exosomal RNA sequencing to identify candidate exosomal miRNAs that could be associated with colorectal cancer (CRC) and its distant metastasis. The expression profiles of exosomal miRNA, as secreted by isogenic human primary CRC cell line SW480 and highly metastatic cell line SW620, were analyzed and the potential targets related to tumorigenesis and metastatic progression were investigated. We found that 25 miRNAs had been up-regulated and 5 miRNAs had been down-regulated in exosomes purified from SW620 culture supernatant. Candidate miRNAs were further evaluated for CRC diagnosis using quantitative real-time polymerase chain reaction in CRC patients. Higher expression levels of circulating exosomal miR-17-5p and miR-92a-3p were significantly associated with pathologic stages and grades of the CRC patients. CONCLUSIONS: Circulating exosomal miR-17-5p and miR-92a-3p may provide a promising noninvasive prognostic biomarker for primary and metastatic CRC.     

Urolithin A induces cell cycle arrest and apoptosis by inhibiting Bcl-2, increasing p53-p21 proteins and reactive oxygen species production in colorectal cancer cells

Colorectal cancer (CRC) is the second most common gastrointestinal cancer globally. Prevention of tumor cell proliferation and metastasis is vital for prolonging patient survival. Polyphenols provide a wide range of health benefits and prevention from cancer. In the gut, urolithins are the major metabolites of polyphenols. The objective of our study was to elucidate the molecular mechanism of the anticancer effect of urolithin A (UA) on colorectal cancer cells. UA was found to inhibit the cell proliferation of CRC cell lines in a dose-dependent and time-dependent manner in HT29, SW480, and SW620 cells. Exposure to UA resulted in cell cycle arrest in a dose-dependent manner along with alteration in the expression of cell cycle–related protein. Treatment of CRC cell lines with UA resulted in the induction of apoptosis. Treatment of HT29, SW480, and SW620 with UA resulted in increased expression of the pro-apoptotic proteins, p53 and p21. Similarly, UA treatment inhibited the anti-apoptotic protein expression of Bcl-2. Moreover, exposure of UA induced cytochrome c release and caspase activation. Furthermore, UA was found to generate reactive oxygen species (ROS) production in CRC cells. These findings indicate that UA possesses anticancer potential and may be used therapeutically for the treatment of CRC.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12192-020-01189-8.     

20(S)‐Protopanaxadiol inhibits epithelial‐mesenchymal transition by promoting retinoid X receptor alpha in human colorectal carcinoma cells

Colorectal carcinoma (CRC) recurrence is often accompanied by metastasis. Most metastasis undergo through epithelial‐mesenchymal transition (EMT). Studies showed that retinol X receptor alpha (RXRα) and 20(S)‐Protopanaxadiol (PPD) have anti‐tumour effects. However, the anti‐metastasis effect of 20(S)‐PPD and the effect of RXRα on EMT‐induced metastasis are few studies on. Therefore, the role of RXRα and 20(S)‐PPD in CRC cell metastasis remains to be fully elucidated. RXRα with clinicopathological characteristics and EMT‐related expression in clinical samples were examined. Then, RXRα and EMT level in SW480 and SW620 cells, overexpressed and silenced RXRα in SW620 cells and SW480 cells, respectively, were evaluated. Finally, 20(S)‐PPD effect on SW620 and SW480 cells was evaluated. The results showed that a lower RXRα expression in cancer tissues, and a moderate negative correlation between RXRα and N stage, and tended to higher level of EMT. SW480 and SW620 cells had the highest and lowest RXRα expression among four CRC cell lines. SW480 had lower EMT level than SW620. Furthermore, 20(S)‐PPD increased RXRα and inhibited EMT level in SW620 cell. Finally, 20(S)‐PPD cannot restore SW480 cells EMT level to normal when RXRα silencing. These findings suggest that 20(S)‐PPD may inhibit EMT process in CRC cells by regulating RXRα expression.     

Proteomics identification of ITGB3 as a key regulator in reactive oxygen species-induced migration and invasion of colorectal cancer cells

Colorectal cancer (CRC) is the third most commonly diagnosed cancer in males and second in females worldwide. Unfortunately 40-50% of patients already have metastatic disease at presentation when prognosis is poor with a 5-year survival of <10%. Reactive oxygen species (ROS) have been proposed to play a crucial role in tumor metastasis. We now show that higher levels of ROS accumulation are found in a colorectal cancer-derived metastatic cell line (SW620) compared with a cell line (SW480) derived from the primary lesion from the same patient. In addition, ROS accumulation can affect both the migratory and invasive capacity of SW480 and SW620 cells. To explore the molecular mechanism underlying ROS-induced migration and invasion in CRC, we have compared protein expression patterns between SW480 and SW620 cells using a two-dimensional electrophoresis-based proteomics strategy. A total of 63 altered proteins were identified from tandem MS analysis. Cluster analysis revealed dysregulated expression of multiple redox regulative or ROS responsive proteins, implicating their functional roles in colorectal cancer metastasis. Molecular and pathological validation demonstrated that altered expression of PGAM1, GRB2, DJ-1, ITGB3, SOD-1, and STMN1 was closely correlated with the metastatic potential of CRC. Functional studies showed that ROS markedly up-regulated expression of ITGB3, which in turn promoted an aggressive phenotype in SW480 cells, with concomitant up-regulated expression of STMN1. In contrast, knockdown of ITGB3 expression could mitigate the migratory and invasive potential of SW620 or H(2)O(2)-treated SW480 cells, accompanied by down-regulated expression of STMN1. The function of ITGB3 was dependent on the surface expression of integrin αvβ3 heterodimer. Furthermore, STMN1 expression and the PI3K-Akt-mTOR pathway were found to be involved in ROS-induced and ITGB3-mediated migration and invasion of colorectal cancer cells. Taken together, these studies suggest that ITGB3 plays an important role in ROS-induced migration and invasion in CRC.     

Silibinin triggers apoptotic signaling pathways and autophagic survival response in human colon adenocarcinoma cells and their derived metastatic cells

Silibinin, a flavonolignan isolated from the milk thistle plant (Silybum marianum), possesses anti-neoplastic properties. In vitro and in vivo studies have recently shown that silibinin inhibits the growth of colorectal cancer (CRC). The present study investigates the mechanisms of silibinin-induced cell death using an in vitro model of human colon cancer progression, consisting of primary tumor cells (SW480) and their derived metastatic cells (SW620) isolated from a metastasis of the same patient. Silibinin induced apoptotic cell death evidenced by DNA fragmentation and activation of caspase-3 in both cell lines. Silibinin enhanced the expression (protein and mRNA) of TNF-related apoptosis-inducing ligand (TRAIL) death receptors (DR4/DR5) at the cell surface in SW480 cells, and induced their expression in TRAIL-resistant SW620 cells normally not expressing DR4/DR5. Caspase-8 and -10 were activated demonstrating the involvement of the extrinsic apoptotic pathway in silibinin-treated SW480 and SW620 cells. The protein Bid was cleaved in SW480 cells indicating a cross-talk between extrinsic and intrinsic apoptotic pathway. We demonstrated that silibinin activated also the intrinsic apoptotic pathway in both cell lines, including the perturbation of the mitochondrial membrane potential, the release of cytochrome c into the cytosol and the activation of caspase-9. Simultaneously to apoptosis, silibinin triggered an autophagic response. The inhibition of autophagy with a specific inhibitor enhanced cell death, suggesting a cytoprotective function for autophagy in silibinin-treated cells. Taken together, our data show that silibinin initiated in SW480 and SW620 cells an autophagic-mediated survival response overwhelmed by the activation of both the extrinsic and intrinsic apoptotic pathways.     

Deep Sequencing of RNA from Three Different Extracellular Vesicle (EV) Subtypes Released from the Human LIM1863 Colon Cancer Cell Line Uncovers Distinct Mirna-Enrichment Signatures

Secreted microRNAs (miRNAs) enclosed within extracellular vesicles (EVs) play a pivotal role in intercellular communication by regulating recipient cell gene expression and affecting target cell function. Here, we report the isolation of three distinct EV subtypes from the human colon carcinoma cell line LIM1863 – shed microvesicles (sMVs) and two exosome populations (immunoaffinity isolated A33-exosomes and EpCAM-exosomes). Deep sequencing of miRNA libraries prepared from parental LIM1863 cells/derived EV subtype RNA yielded 254 miRNA identifications, of which 63 are selectively enriched in the EVs - miR-19a/b-3p, miR-378a/c/d, and miR-577 and members of the let-7 and miR-8 families being the most prominent. Let-7a-3p*, let-7f-1-3p*, miR-451a, miR-574-5p*, miR-4454 and miR-7641 are common to all EV subtypes, and 6 miRNAs (miR-320a/b/c/d, miR-221-3p, and miR-200c-3p) discern LIM1863 exosomes from sMVs; miR-98-5p was selectively represented only in sMVs. Notably, A33-Exos contained the largest number (32) of exclusively-enriched miRNAs; 14 of these miRNAs have not been reported in the context of CRC tissue/biofluid analyses and warrant further examination as potential diagnostic markers of CRC. Surprisingly, miRNA passenger strands (star miRNAs) for miR-3613-3p*, -362-3p*, -625-3p*, -6842-3p* were the dominant strand in A33-Exos, the converse to that observed in parental cells. This finding suggests miRNA biogenesis may be interlinked with endosomal/exosomal processing.     

Cytotoxic effects of the polyamine oxidase inactivator MDL 72527 to two human colon carcinoma cell lines SW480 and SW620

N ¹,N ⁴-bis(2,3-butadienyl)-1,4-butanediamine (MDL 72527) was considered to be a selective inactivator of FAD-dependent tissue polyamine oxidase. Recently MDL 72527 was reported to induce apoptosis in transformed hematopoietic cells through lysosomotropic effects. Since it is the only useful inhibitor of polyamine oxidase available at present, the re-evaluation of its properties seemed important. Human colon carcinoma-derived SW480 cells and their lymph node metastatic derivatives (SW620) were chosen for our study because they differ in various aspects of polyamine metabolism but have similar polyamine oxidase activities. MDL 72527 inhibited cell growth in a concentration-dependent manner, depleted intracellular polyamine pools, and caused the accumulation of N ¹-acetyl derivatives of spermidine and spermine. SW620 cells were more sensitive to the drug than were SW480 cells. At 150 μmol/L MDL 72527, SW620 cells accumulated in S-phase of the cell cycle, showed decreased polyamine transport rate, and showed no increase of polyamine N ¹-acetyltransferase activity. In contrast, SW480 cells were not arrested in a particular phase of the cell cycle, showed enhanced polyamine uptake, and showed a mild induction of acetyltransferase. The results suggest that MDL 72527 retains its value as a selective tool in short-term experiments only at concentrations not exceeding those necessary for the inactivation of polyamine oxidase. At concentrations above 50 μmol/L and at exposure times longer than 24 h, it may derange cell functions nonspecifically, and thus blur the results of studies intended to elucidate polyamine oxidase functions. This revised version was published online in July 2006 with corrections to the Cover Date.     

Annexin A3 depletion overcomes resistance to oxaliplatin in colorectal cancer via the MAPK signaling pathway

Colorectal cancer (CRC) is a common disease with high mortality and morbidity. Annexin A3 (ANXA3) belongs to the structurally homologous family of Ca²⁺ and phospholipid-binding proteins. This study aimed to investigate the effects and potential mechanisms of ANXA3 on oxaliplatin (Ox) resistance in CRC. We generated two human CRC cell lines (HCT116/Ox and SW480/Ox) with acquired Ox resistance and determined their resistance properties. ANXA3 expression and cell apoptosis, migration and invasion also were evaluated. We found that cell viability of HCT116/Ox and SW480/Ox was higher than that in parental cells in the presence of Ox. ANXA3 was highly expressed in HCT116/Ox and SW480/Ox cells. ANXA3 downregulation diminished cell survival, migration and invasion, while increased the apoptosis of HCT116 and SW480 with or without Ox. Moreover, depletion of ANXA3 reduced cell viability and BrdU incorporation, increased cell apoptosis and c-caspase 3 expression in HCT116/Ox with or without Ox. A transwell assay determined that knockdown of ANXA3 impeded the migration and invasion of HCT116/Ox and SW480/Ox cells. Additionally, phosphorylation of extracellular signal–regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) decreased upon ANXA3 depletion in HCT116/Ox cells, and ANXA3 silencing suppressed Ox-induced activation of ERK and JNK signaling pathway. ANXA3 downregulation reduced Ox resistance in CRC, and treatment with the ERK inhibitor PD098059 or JNK inhibitor SP600125 contributed to this process. These results indicate that silencing ANXA3 could overcome Ox resistance in CRC via the mitogen-activated protein kinase signaling pathway.     

Regulatory effects of lncRNA ATB targeting miR-200c on proliferation and apoptosis of colorectal cancer cells

This investigation was intended to elucidate whether long noncoding RNA (lncRNA)-activated by transforming growth factor-β (ATB) interacting with miR-200c could mediate colorectal cancer (CRC) progression, offering potential strategies for diagnosing and treating CRC. Here totally 315 patients with CRC were recruited, and their CRC tissues and adjacent normal tissues were gathered. Concurrently, four colon cancer cell lines (ie, SW620, Lovo, HCT116, and SW480) and the human colon mucosal epithelial cell line (NCM460) were also purchased. Moreover, si-ATB, si-NC, miR-200c mimic, miR-200c inhibitor, and miR-NC were prepared for transfection into the CRC cells, and their effects on CRC cell lines were evaluated based on the conduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, colony formation assay, and flow cytometry assay. Eventually, the Luciferase reporter gene assay was carried out to judge if there existed a targeted relationship between ATB and miR-200c. The results of Cox regression analyses suggested that overexpressed lncRNA ATB, underexpressed miR-200c, poor tumor differentiation, lymph-vascular invasion, and perineural invasion were symbolic of shortened survival of the patients with CRC (all P < .05). Besides, transfection of pcDNA3.1-ATB and miR-200c inhibitor could boost the viability and proliferation of Lovo and SW620 cell lines (all P < .05). Meanwhile, the expressions of p53 and p21 were also reduced under treatments of pcDNA3.1-ATB and miR-200c inhibitor (P < .05). In addition, CDK2 seemed to reverse the contribution of miR-200c to intensifying viability and proliferation of Lovo and SW420 cell lines (P < .05). Furthermore, ATB might downregulate miR-200c expression by targeting it (P < .05), and CDK2 was subjected to dual regulation of both ATB and miR-200c (P < .05). In conclusion, the lncRNA ATB/miR-200c/CDK2 signaling was responsible for intensified proliferation and prohibited apoptosis of CRC cells, which might provide effective approaches for diagnosing and treating CRC.     

The monoclonal antibody CJA3 down-regulates the susceptibility of human tumor cell lines to natural cell-mediated cytotoxicity

While developing monoclonal antibodies (MoAb) to colorectal carcinoma (CRC) cells, we noted that one MoAb, termed CJA3, down-regulated natural cell-mediated cytotoxicity (NCMC) against CRC cell lines SW480 and SW620. The MoAb CJA3 was developed by immunizing a BALB/c mouse with fresh human colorectal adenocarcinoma cells. The antigen recognized by the MoAb CJA3 was expressed on several solid tumor cell lines and on one of the six lymphoreticular cell lines tested, but was not detected on normal peripheral blood lymphocytes (PBL). SDS-PAGE analysis of the antigen immunoprecipitated by the MoAb CJA3 from the CRC cell lines SW480 and SW620 and from the melanoma cell line MALME-3M revealed a component with a m.w. of 150,000. Preincubation of CRC cell lines SW480 and SW620 with the MoAb CJA3 for 16 hr reduced their susceptibility to NCMC by about 50%. Kinetic experiments showed that prolongation of the incubation of target cells with the MoAb CJA3 resulted in a time-dependent increase in the amount of MoAb bound. Maximum binding of the MoAb CJA3 was reached after 4 hr of incubation. The increase in antigen expression chronologically paralleled the decrease in NCMC target cell sensitivity, suggesting that the membrane alterations induced by the MoAb CJA3 were important for NCMC against these two cell lines.     

Near-infrared fluorescence-labeled anti-PD-L1-mAb for tumor imaging in human colorectal cancer xenografted mice

The expression of programmed death ligand-1 (PD-L1) in tumor has been used as a biomarker to predict the anti-PD-L1 immunotherapy response. To develop a noninvasive imaging technique to monitor the dynamic changes in PD-L1 expression in colorectal cancer (CRC), we labeled an anti-PD-L1 monoclonal antibody with near-infrared (NIR) dye and tested the ability of the NIR-PD-L1-mAb probe to monitor the PD-L1 expression in CRC-xenografted mice by performing optical imaging. Consistent with the expression levels of PD-L1 protein in three CRC cell lines in vitro by flow cytometry and Western blot analyses, our in vivo imaging showed the highest fluorescence signal of the xenografted tumors in mice bearing SW620 CRC cells, followed by tumors derived from SW480 and HCT8 cell lines. We detected the highest fluorescent intensity of the tumor at 120 hours after injection of NIR-PD-L1-mAb. The highest fluorescence intensity was seen in the tumor, followed by the spleen and the liver in SW620 xenografted mice. In SW480 and HCT8 xenografted mice, however, the highest fluorescent signals were detected in the spleen, followed by the liver and the tumor. Our findings indicate that SW620 cells express a higher level of PD-L1, and the NIR-PD-L1-mAb binding to PD-L1 on the surface of CRC cells was specific. The technique was safe and could provide valuable information on PD-L1 expression of the tumor for development of a therapeutic strategy of personized targeted immunotherapies as well as treatment response of patients with CRC.     

Endotoxin/lipopolysaccharide activates NF-kappa B and enhances tumor cell adhesion and invasion through a beta 1 integrin-dependent mechanism

Beta(1) integrins play a crucial role in supporting tumor cell attachment to and invasion into the extracellular matrix. Endotoxin/LPS introduced by surgery has been shown to enhance tumor metastasis in a murine model. Here we show the direct effect of LPS on tumor cell adhesion and invasion in extracellular matrix proteins through a beta(1) integrin-dependent pathway. The human colorectal tumor cell lines SW480 and SW620 constitutively expressed high levels of the beta(1) subunit, whereas various low levels of alpha(1), alpha(2), alpha(4), and alpha(6) expression were detected. SW480 and SW620 did not express membrane-bound CD14; however, LPS in the presence of soluble CD14 (sCD14) significantly up-regulated beta(1) integrin expression; enhanced tumor cell attachment to fibronectin, collagen I, and laminin; and strongly promoted tumor cell invasion through the Matrigel. Anti-beta(1) blocking mAbs (4B4 and 6S6) abrogated LPS- plus sCD14-induced tumor cell adhesion and invasion. Furthermore, LPS, when combined with sCD14, resulted in NF-kappaB activation in both SW480 and SW620 cells. Inhibition of the NF-kappaB pathway significantly attenuated LPS-induced up-regulation of beta(1) integrin expression and prevented tumor cell adhesion and invasion. These results provide direct evidence that although SW480 and SW620 cells do not express membrane-bound CD14, LPS in the presence of sCD14 can activate NF-kappaB, up-regulate beta(1) integrin expression, and subsequently promote tumor cell adhesion and invasion. Moreover, LPS-induced tumor cell attachment to and invasion through extracellular matrix proteins is beta(1) subunit-dependent.     

Dual inhibition of BDNF/TrkB and autophagy: a promising therapeutic approach for colorectal cancer

Colorectal cancer (CRC) is the most common digestive cancer in the Western world. Despite effective therapies, resistance and/or recurrence frequently occur. The present study investigated the impact of two survival pathways—neurotrophic factors (TrkB/BDNF) and autophagy—on cell fate and tumour evolution. In vitro studies were performed on two CRC cell lines, SW480 (primary tumour) and SW620 (lymph node invasion), which were also used for subcutaneous xenografts on a nude mouse model. In addition, the presence of neurotrophic factors (NTs) and autophagy markers were assessed in tissue samples representative of different stages. On the basis of our previous study (which demonstrated that TrkB overexpression is associated with prosurvival signaling in CRC cells), we pharmacologically inhibited NTs pathways with K252a. As expected, an inactivation of the PI3K/AKT pathway was observed and CRC cells initiated autophagy. Conversely, blocking the autophagic flux with chloroquine or with ATG5‐siRNA overactivated TrkB/BDNF signaling. In vitro, dual inhibition improved the effectiveness of single treatment by significantly reducing metabolic activity and enhancing apoptotic cell death. These findings were accentuated in vivo, in which dual inhibition induced a spectacular reduction in tumour volume following long‐term treatment (21 days for K252a and 12 days for CQ). Finally, significant amounts of phospho‐TrkB and LC3II were found in the patients’ tissues, highlighting their relevance in CRC tumour biology. Taken together, our results show that targeting NTs and autophagy pathways potentially constitutes a new therapeutic approach for CRC.     

Vimentin,colon cancer progression and resistance to butyrate and other HDACis

Dietary fibre protects against colorectal cancer (CRC) most likely through the activity of its fermentation product, butyrate. Butyrate functions as a histone deacetylase inhibitor (HDACi) that hyperactivates Wnt signalling and induces apoptosis of CRC cells. However, individuals who consume a high‐fibre diet may still develop CRC; therefore, butyrate resistance may develop over time. Furthermore, CRC cells that are resistant to butyrate are cross‐resistant to clinically relevant therapeutic HDACis, suggesting that the development of butyrate resistance in vivo can result in HDACi‐resistant CRCs. Butyrate/HDACi‐resistant CRC cells differ from their butyrate/HDACi‐sensitive counterparts in the expression of many genes, including the gene encoding vimentin (VIM) that is usually expressed in normal mesenchymal cells and is involved in cancer metastasis. Interestingly, vimentin is overexpressed in butyrate/HDACi‐resistant CRC cells although Wnt signalling is suppressed in such cells and that VIM is a Wnt activity‐targeted gene. The expression of vimentin in colonic neoplastic cells could be correlated with the stage of neoplastic progression. For example, comparative analyses of LT97 microadenoma cells and SW620 colon carcinoma cells revealed that although vimentin is not detectable in LT97 cells, it is highly expressed in SW620 cells. Based upon these observations, we propose that the differential expression of vimentin contributes to the phenotypic differences between butyrate‐resistant and butyrate‐sensitive CRC cells, as well as to the differences between early‐stage and metastatic colorectal neoplastic cells. We discuss the hypothesis that vimentin is a key factor integrating epithelial to mesenchymal transition, colonic neoplastic progression and resistance to HDACis.