Senescence In Colorectal Cancer Cells Research Articles

Therapy-induced senescent tumor cell-derived extracellular vesicles promote colorectal cancer progression through SERPINE1-mediated NF-κB p65 nuclear translocation

BackgroundCellular senescence frequently occurs during anti-cancer treatment, and persistent senescent tumor cells (STCs) unfavorably promote tumor progression through paracrine secretion of the senescence-associated secretory phenotype (SASP). Extracellular vesicles (EVs) have recently emerged as a novel component of the SASP and primarily mediate the tumor-promoting effect of the SASP. Of note, the potential effect of EVs released from STCs on tumor progression remains largely unknown.MethodsWe collected tumor tissues from two cohorts of colorectal cancer (CRC) patients to examine the expression of p16, p21, and SERPINE1 before and after anti-cancer treatment. Cohort 1 included 22 patients with locally advanced rectal cancer (LARC) who received neoadjuvant therapy before surgical resection. Cohort 2 included 30 patients with metastatic CRC (mCRC) who received first-line irinotecan-contained treatment. CCK-8, transwell, wound-healing assay, and tumor xenograft experiments were carried out to determine the impacts of EVs released from STCs on CRC progression in vitro and in vivo. Quantitative proteomic analysis was applied to identify protein cargo inside EVs secreted from STCs. Immunoprecipitation and mass spectrometer identification were utilized to explore the binding partners of SERPINE1. The interaction of SERPINE1 with p65 was verified by co-immunoprecipitation, and their co-localization was confirmed by immunofluorescence.ResultsChemotherapeutic agents and irradiation could potently induce senescence in CRC cells in vitro and in human CRC tissues. The more significant elevation of p16 and p21 expression in patients after anti-cancer treatment displayed shorter disease-free survival (DFS) for LARC or progression-free survival (PFS) for mCRC. We observed that compared to non-STCs, STCs released an increased number of EVs enriched in SERPINE1, which further promoted the progression of recipient cancer cells. Targeting SERPINE1 with a specific inhibitor, tiplaxtinin, markedly attenuated the tumor-promoting effect of STCs-derived EVs. Additionally, the patients with greater increment of SERPINE1 expression after anti-cancer treatment had shorter DFS for LARC or PFS for mCRC. Mechanistically, SERPINE1 bound to p65, promoting its nuclear translocation and subsequently activating the NF-κB signaling pathway.ConclusionsWe provide the in vivo evidence of the clinical prognostic implications of therapy-induced senescence. Our results revealed that STCs were responsible for CRC progression by producing large amounts of EVs enriched in SERPINE1. These findings further confirm the crucial role of therapy-induced senescence in tumor progression and offer a potential therapeutic strategy for CRC treatment.

Cellular senescence gene TACC3 associated with colorectal cancer risk via genetic and DNA methylated alteration.

Cell senescence genes play a vital role in the pathogenesis of colorectal cancer, a process that may involve the triggering of genetic variations and reversible phenotypes caused by epigenetic modifications. However, the specific regulatory mechanisms remain unclear. Using CellAge and The Cancer Genome Atlas databases and in-house RNA-seq data, DNA methylation-modified cellular senescence genes (DMCSGs) were validated by Support Vector Machine and correlation analyses. In 1150 cases and 1342 controls, we identified colorectal cancer risk variants in DMCSGs. The regulatory effects of gene, variant, and DNA methylation were explored through dual-luciferase and 5-azacytidine treatment experiments, complemented by multiple database analyses. Biological functions of key gene were evaluated via cell proliferation assays, SA-β-gal staining, senescence marker detection, and immune infiltration analyses. The genetic variant rs4558926 in the downstream of TACC3 was significantly associated with colorectal cancer risk (OR=1.35, P=3.22×10-4). TACC3 mRNA expression increased due to rs4558926 C>G and decreased DNA methylation levels. The CpG sites in the TACC3 promoter region were regulated by rs4558926. TACC3 knockdown decreased proliferation and senescence in colorectal cancer cells. In addition, subjects with high-TACC3 expression presented an immunosuppressive microenvironment. These findings provide insights into the involvement of genetic variants of cellular senescence genes in the development and progression of colorectal cancer.

Avenanthramide C induces cellular senescence in colorectal cancer cells via suppressing β-catenin-mediated the transcription of miR-183/96/182 cluster

Cellular senescence is representing a potential anticancer therapeutic arsenal. Avenanthramide C (AVN C), as a signature compound of oats, exhibits antioxidant, anti-inflammatory, anti-atherosclerotic, and anti-tumor activities. However, the relationship between AVN C and cellular senescence in tumors remains largely unclear. Here, we elucidated that AVN C treatment predisposed colorectal cancer cells to senescent phenotype confirmed by flattened and enlarged shape characteristics, elevated senescence-associated β-galactosidase (SA-β-Gal) activity, and G1 phase arrest. Furthermore, AVN C triggered cellular senescence via transcriptionally repressing miR-183/96/182 cluster and subsequently reduced the levels of mature miR-183, −96, and −182. Mechanistically, AVN C exerted its senescence induction by attenuating β-catenin-mediated transactivation of miR-183/96/182 cluster to unleash its common target FOXO1 and two other targets, FOXO3 and SMAD4, which subsequently foster the p21 and p16 expression. In addition, AVN C is also noted to facilitate p53-mediated p21 transactivation via suppressing β-catenin. Collectively, we identified a novel mechanism of β-catenin/miR-183/96/182 cluster/FOXO1 mediated-CRC cellular senescence that entails that AVN C serves as an auxiliary agent for CRC treatment.

Pyrimethamine inhibits cell growth by inducing cell senescence and boosting CD8+ T-cell mediated cytotoxicity in colorectal cancer

BackgroundThe emergence of nonresponse or resistance to traditional chemotherapeutic agents is one of the main challenges of colorectal cancer (CRC) therapies. Thus, novel therapeutic drugs that can improve the clinical outcomes of CRC patients are urgently needed. The purpose of this study was to investigate the effects and mechanisms of pyrimethamine in CRC.Methods and resultsIn this study, we assessed the role of pyrimethamine on CRC cell growth by cell counting kit-8 and colony formation assays. Cell cycle distribution and cellular senescence were determined by flow cytometry and senescence-associated β-galactosidase staining respectively. RNA-seq analysis and western blotting were used to investigate the potential pathways of pyrimethamine in CRC development. Moreover, animal experiments were performed to evaluate the effect of pyrimethamine in vivo. Our results demonstrated that pyrimethamine could inhibit cell growth by inducing S phase arrest followed by cellular senescence in CRC cells, and the p38MAPK-p53 axis was probably involved in that effect. In addition, pyrimethamine could also boost CD8+ T-cell mediated cytotoxicity and exert antitumor activity in vivo.ConclusionThese results indicated that pyrimethamine may be a promising candidate agent for CRC treatment.

Cucurbitacin E Triggers Cellular Senescence in Colon Cancer Cells via Regulating the miR-371b-5p/TFAP4 Signaling Pathway.

The induction of cellular senescence is considered as a potent strategy to suppress cancer progression. Cucurbitacin E (CE) belongs to the triterpenoids and has received substantial attention for its antineoplastic property. However, the function of CE on cellular senescence remained elusive. Herein, we revealed that CE significantly induced cellular senescence in colorectal cancer (CRC) cells. The CE effects on the cellular senescence in CRC cells were confirmed by observing the common features of the senescence, such as the enhanced activity of senescence-associated β-galactosidase, γ-H2AX positive staining, and upregulation of senescence-associated proteins including p53, p27, and p21. Moreover, CE exerted pro-senescent effects in CRC cells via attenuating the transcription factor activating enhancer-binding protein 4 (TFAP4) expression, and the ectopic expression of TFAP4 blocked the CE-induced senescence. Mechanistically, CE treatment caused a robust increase in miR-371b-5p, which markedly repressed TFAP4. In contrast, silencing of miR-371b-5p counteracted the percentages of CE-induced senescent cells from 37.49 ± 2.61 to 7.06 ± 0.91% in HCT-116 cells via derepressing TFAP4 to attenuate the expression of p53, p21, and p16. Altogether, these results demonstrated that dietary CE induces CRC cellular senescence via modulating the miR-371b-5p/TFAP4 axis and presents opportunities for potential therapeutic strategies against CRC.

Predicting survival and immune microenvironment in colorectal cancer: a STAT signaling-related signature.

Despite research advances, studies on predictive models of colorectal cancer (CRC) remain scarce and none have evaluated signal transducer and activator of transcription (STAT) signaling. To develop an effective prognostic signature for and evaluate its association with immune microenvironment. Comprehensive analysis based on The Cancer Genome Atlas and Gene Expression Omnibus databases with experimental validation. Gene expression and clinical profiles of CRC patients were extracted from the databases. Differentially expressed genes with prognostic values were used to construct a signature. Immune cell infiltration and composition were further evaluated by TIMER, single-sample gene set enrichment and CIBERSORT analyses. The impact of the hub gene Caveolin-1 (CAV1) on cell proliferation, apoptosis, senescence and tumor angiogenesis was experimentally validated. The five-gene-based STAT signaling-related prognostic signature was significantly associated with CRC survival, and the nomogram was with improved prognostic efficacy than the conventional TNM stage. The STAT signaling-related signature was correlated with tumor immune microenvironment. CAV1 was further identified as the hub gene within the signature. CAV1 inhibits the proliferation and induces the apoptosis as well as senescence of CRC cells. In addition, the tumor angiogenesis of CRC can be suppressed by CAV1 overexpression. The STAT signaling-related signature effectively predicts the prognosis and regulates tumor immune microenvironment in CRC. Our study underscores the role of STAT regulator, CAV1, as an important tumor suppressor in CRC carcinogenesis. Modulating STAT and its regulators could be a promising strategy for CRC in clinical practice.

Targeting NUFIP1 Suppresses Growth and Induces Senescence of Colorectal Cancer Cells.

NUFIP1 is an RNA-binding protein that interacts with fragile X mental retardation protein (FMRP) in the messenger ribonucleoprotein particle (mRNP). We previously showed that NUFIP1 was upregulated in colorectal cancer (CRC), but how the protein may contribute to the disease and patient prognosis is unknown. Here we combine database analysis, microarray, quantitative PCR, and immunohistochemistry of patients’ samples to confirm our previous findings on NUFIP1 overexpression in CRC, and to reveal that increased expression of NUFIP1 in CRC tissues correlated with worse overall, recurrence-free, event-free and disease-free survival in patients, as well as with more advanced CRC clinicopathological stage. Loss of function analysis demonstrated that NUFIP1 knockdown suppressed cell growth in vitro and in vivo, inhibited cell viability and survival, and induced cell cycle arrest and apoptosis in vitro, as well as up-regulated Bax and down-regulated Bcl-2 protein expression. In addition, as a natural anticancer triterpene from various fruits and vegetables, ursolic acid (UA) treatment suppressed cell proliferation, down-regulated NUFIP1 protein expression, and further enhanced the effects of NUFIP1 knockdown in CRC cells in vitro. NUFIP1 knockdown up-regulated the expression of 136 proteins, down-regulated the expression of 41 proteins, and enriched multiple signaling pathways including the senescence-associated heterochromatin foci (SAHF) pathway. Furthermore, NUFIP1 knockdown enhanced the expression of senescence-associated-β-galactosidase (SA-β-gal), the SAHF markers HP1γ and trimethylation (H3k9me3), and the senescence-related protein HMGA2, as well as both p53 and its downstream p21 protein expression. Our findings suggest that NUFIP1 is overexpressed in CRC and correlates with disease progression and poor patient survival. NUFIP1 may exert oncogenic effects partly by altering senescence. UA may show potential to treat CRC by down-regulating NUFIP1.

B7-H3 suppresses doxorubicin-induced senescence-like growth arrest in colorectal cancer through the AKT/TM4SF1/SIRT1 pathway

Emerging evidence suggests that cellular senescence induced by chemotherapy has been recognized as a new weapon for cancer therapy. This study aimed to research novel functions of B7-H3 in cellular senescence induced by a low dose of doxorubicin (DOX) in colorectal cancer (CRC). Here, our results demonstrated that B7-H3 knockdown promoted, while B7-H3 overexpression inhibited, DOX-induced cellular senescence. B7-H3 knockdown dramatically enhanced the growth arrest of CRC cells after low-dose DOX treatment, but B7-H3 overexpression had the opposite effect. By RNA-seq analysis and western blot, we showed that B7-H3 prevented cellular senescence and growth arrest through the AKT/TM4SF1/SIRT1 pathway. Blocking the AKT/TM4SF1/SIRT1 pathway dramatically reversed B7-H3-induced resistance to cellular senescence. More importantly, B7-H3 inhibited DOX-induced cellular senescence of CRC cells in vivo. Therefore, targeting B7-H3 or the B7-H3/AKT/TM4SF1/SIRT1 pathway might be a new strategy for promoting cellular senescence-like growth arrest during drug treatment in CRC.

Oxaliplatin-Induced Senescence in Colorectal Cancer Cells Depends on p14ARF-Mediated Sustained p53 Activation

Simple SummaryChemotherapy can lead to cellular senescence in tumor cells. Here we demonstrate that oxaliplatin induces senescence in p53-proficient colorectal cancer (CRC) cells and leads to the G2-phase arrest in all lines studied (HCT116p53+/+, HCT116p53−/−, LoVo, SW48, and SW480). At early times the p53-competent lines activate p53 and p21CIP1, however, at later times, only LoVo cells showed sustained p53/p21CIP1 activation, accompanied by a strong induction of senescence and senescence-associated secretory phenotype (SASP) factors. Opposite to LoVo, the p53/p21CIP1 response and senescence induction is much weaker in the other p53-proficient cells, due to p14ARF deficiency. LoVo cells express p14ARF protein and siRNA-mediated knockdown of p14ARF significantly reduces sustained p53/p21CIP1 activation and senescence. Vice versa, ectopic expression of p14ARF enhances oxaliplatin-induced senescence in SW48 and SW480 cells. Our data show that oxaliplatin-induced senescence in CRC cells depends on p53 proficiency; however, a significant induction can only be observed upon p14ARF-mediated p53 stabilization.Senescence is an important consequence of cytostatic drug-based tumor therapy. Here we analyzed to which degree the anticancer drug oxaliplatin induces cell death, cell cycle arrest, and senescence in colorectal cancer (CRC) cells and elucidated the role of p53. Oxaliplatin treatment resulted in the G2-phase arrest in all CRC lines tested (HCT116p53+/+, HCT116p53−/−, LoVo, SW48 and SW480). Immunoblot analysis showed that within the p53-competent lines p53 and p21CIP1 are activated at early times upon oxaliplatin treatment. However, at later times, only LoVo cells showed sustained activation of the p53/p21CIP1 pathway, accompanied by a strong induction of senescence as measured by senescence-associated β-Gal staining and induction of senescence-associated secretory phenotype (SASP) factors. Opposite to LoVo, the p53/p21CIP1 response and senescence induction was much weaker in the p53-proficient SW48 and SW480 cells, which was due to deficiency for p14ARF. Thus, among lines studied only LoVo express p14ARF protein and siRNA-mediated knockdown of p14ARF significantly reduced sustained p53/p21CIP1 activation and senescence. Vice versa, ectopic p14ARF expression enhanced oxaliplatin-induced senescence in SW48 and SW480 cells. Our data show that oxaliplatin-induced senescence in CRC cells is dependent on p53 proficiency; however, a significant induction can only be observed upon p14ARF-mediated p53 stabilization.

PIK3R3 inhibits cell senescence through p53/p21 signaling

Cellular senescence is a stress response of human cells that removes potentially harmful cells by initiating cell cycle arrest. Inducing senescence of tumor cells may be an effective tumor-inhibiting strategy. In this study we found that PIK3R3 could inhibit the cell senescence of colorectal cancer cells and promote cell proliferation through the p53/p21 signal pathway. PIK3R3 could bind to p53 and inhibit the binding of p53 to the p21 gene promoter region, and thus affecting the transcriptional activity of p21 gene. Our study has provided new evidence of the role of PIK3R3 in p53 regulation and inhibition of PIK3R3 may be one of the potential targets of tumor therapy.

N-Acetyltransferase 10 Promotes Micronuclei Formation to Activate the Senescence-Associated Secretory Phenotype Machinery in Colorectal Cancer Cells

The formation of micronuclei (MN) is prevalent in human cancer cells and its role in activating the senescence-associated secretory phenotype (SASP) machinery has been identified recently. However, the role of MN in regulation of SASP signaling still needs to define in practical cancers. Here, we reported that in colorectal cancer cells the expression of NAT10 (N-acetyltransferase 10) could mediate MN formation through DNA replication and NAT10-positive MN could activate SASP by binding to cGAS. The chemical inhibition of NAT10 by Remodelin or genomic depletion could markedly reduce MN formation, SASP activation, and senescence in colorectal cancer cells. Cell stress such as oxidative or hypoxia could upregulate NAT10 and its associated MN formation senescence and expression of SASP factors. Statistical analysis of clinical specimens revealed correlations between NAT10 expression, MN formation, SASP signaling, and the clinicopathological features of colorectal cancer. Our data suggest that NAT10 increasing MN formation and SASP pathway activation, promoting colorectal cancer progression.

Fibrinogen Activates FAK to Promote the Colorectal Adenocarcinoma Growth

Along with other proteins in the coagulation cascade, including tissue factor and thrombin, fibrinogen has been shown to promote tumor metastasis across numerous cancer types. However, the role of fibrin(ogen) in primary tumor growth is context dependent and not universally important. One cancer strongly dependent on fibrin(ogen) for primary tumor growth is colorectal cancer (CRC), but the mechanisms by which fibrinogen supports colon cancer growth are not well understood. To delineate the mechanism of fibrin(ogen)-supported tumor growth, we implanted C57BL/6-derived murine colon cancer cells (MC38) into the dorsal subcutis of syngeneic mice with specific deletions/alteration in fibrinogen or FXIII-deficiency. Complete fibrinogen deficiency significantly limited the growth of colon cancer cells. However, the imposition of FXIII-deficiency or specific mutations in fibrinogen that 1) prevent the formation of fibrin polymer (FibAEK), limit fibrin(ogen) binding to the integrin αMβ2 (Fibɣ390-396A), or remove the ɣ chain binding motif for the platelet integrin αIIbβ3 (FibɣΔ5) had no impact on colon cancer growth. To explore the mechanisms coupling fibrin(ogen) to colon cancer growth using a nonbiased approach, we performed RNA-Seq analyses of murine CRC tumors harvested from Fib+ and Fib- mice. We detected significant differences in the expression of 214 genes (128 downregulated and 86 upregulated) involved in cellular proliferation, survival, migration and metabolism. Notably, Stratifin (SFN), which encodes 14-3-3-σ, was one of the genes found to be highly upregulated in tumors from Fib- mice relative to Fib+ mice. 14-3-3-σ is a potent cell cycle regulator and it is also known to stabilize p53, which ultimately inhibits tumor growth. In a separate validation cohort, we observed significantly increased protein expression of 14-3-3-σ and its upstream and downstream targets (i.e., p53, p21 and p27) in tumors harvested from Fib- mice relative to controls. We also compared FAK activation, a key negative regulator of p53, in tumors from Fib+ and Fib- mice. FAK was inactive in tumors from Fib- mice, as indicated by lack of phosphorylation at tyrosine 397 (p-FAK Tyr397). Also, MDM2 was less phosphorylated at Ser166 in Fib- tumors, suggesting that p53 is not degraded by MDM2 in the absence of fibrinogen. Taken together, these data suggest that fibrin(ogen)-mediated downregulation of p53 and other targets via FAK activation and downregulation of 14-3-3-σ results in senescence of CRC cells. Consistent with this view, Ki67 positive nuclei were significantly less in tumor from Fib- mice relative to controls We also observed senescence-associated-β-galactosidase (SA-β-gal) activity in the tumors from Fib- mice, but not those from Fib+ animals. Furthermore, NMR-based metabolomics analyses demonstrated significantly less NAD+ and lactate levels in tumors from Fib- mice, which further confirms that fibrinogen deficiency hampers proliferation by inhibiting major metabolic pathways. In order to determine if fibrinogen-mediated support of CRC growth is tumor cell intrinsic, we compared MC38 growth in the 3D bioprinted matrices consisting of fibrin or albumin. MC38 cells showed a higher proliferation rate in the fibrinogen printed 3D environment compared to the albumin environment. These findings suggest that fibrin(ogen)-mediated engements of tumor cells activates FAK which inhibits p53 and its downstream targets including 14-3-3-σ and p21 which promote cellular proliferation and prevent senescence. Overall, these studies suggest that fibrin(ogen) is the important component of the CRC microenvironment and could be exploited for targeting and treating the CRC. Disclosures Whitlock: POSNA: Other: Research Committee; MTF Biologics Grant Program: Other: site co-investigator. Palumbo:Ionis Pharmaceuticals: Research Funding.

TRIB2 functions as novel oncogene in colorectal cancer by blocking cellular senescence through AP4/p21 signaling

BackgroundCellular senescence is a state of irreversible cell growth arrest and senescence cells permanently lose proliferation potential. Induction of cellular senescence might be a novel therapy for cancer cells. TRIB2 has been reported to participate in regulating proliferation and drug resistance of various cancer cells. However, the role of TRIB2 in cellular senescence of colorectal cancer (CRC) and its molecular mechanism remains unclear.MethodsThe expression of TRIB2 in colorectal cancer tissues and adjacent tissues was detected by immunohistochemistry and RT-PCR. The growth, cell cycle distribution and cellular senescence of colorectal cancer cells were evaluated by Cell Counting Kit-8 (CCK8) assay, flow cytometry detection and senescence-associated β-galactosidase staining, respectively. Western blot, RT-PCR and luciferase assay were performed to determine how TRIB2 regulates p21. Immunoprecipitation (IP) and chromatin-immunoprecipitation (ChIP) were used to investigate the molecular mechanisms.ResultsWe found that TRIB2 expression was elevated in CRC tissues compared to normal adjacent tissues and high TRIB2 expression indicated poor prognosis of CRC patients. Functionally, depletion of TRIB2 inhibited cancer cells proliferation, induced cell cycle arrest and promoted cellular senescence, whereas overexpression of TRIB2 accelerated cell growth, cell cycle progression and blocked cellular senescence. Further studies showed that TRIB2 physically interacted with AP4 and inhibited p21 expression through enhancing transcription activities of AP4. The rescue experiments indicated that silencing of AP4 abrogated the inhibition of cellular senescence induced by TRIB2 overexpression.ConclusionThese data demonstrate that TRIB2 suppresses cellular senescence through interaction with AP4 to down-regulate p21 expression. Therefore, TRIB2 could be a potential target for CRC treatment.

Coroglaucigenin induces senescence and autophagy in colorectal cancer cells

Coroglaucigenin (CGN), a natural product isolated from Calotropis gigantean by our research group, has been identified as a potential anti-cancer agent. However, the molecular mechanisms involved remain poorly understood. Cell viability and cell proliferation were detected by MTT and BrdU assays. Flow cytometry, SA-β-gal assay, western blotting and immunofluorescence were performed to determine CGN-induced apoptosis, senescence and autophagy. Western blotting, siRNA transfection and coimmunoprecipitation were carried out to investigate the mechanisms of CGN-induced senescence and autophagy. The anti-tumour activities of combination therapy with CGN and chloroquine were observed in mice tumour models. We demonstrated that CGN inhibits the proliferation of colorectal cancer cells both in vitro and in vivo. We showed that the inhibition of cell proliferation by CGN is independent of apoptosis, but is associated with cell-cycle arrest and senescence in colorectal cancer cells. Notably, CGN induces protective autophagy that attenuates CGN-mediated cell proliferation. Functional studies revealed that CGN disrupts the association of Hsp90 with both CDK4 and Akt, leading to CDK4 degradation and Akt dephosphorylation, eventually resulting in senescence and autophagy, respectively. Combination therapy with CGN and chloroquine resulted in enhanced anti-tumour effects in vivo. Our results demonstrate that CGN induces senescence and autophagy in colorectal cancer cells and indicate that combining it with an autophagy inhibitor may be a novel strategy suitable for CGN-mediated anti-cancer therapy.

Silence of long noncoding RNA PANDAR switches low-dose curcumin-induced senescence to apoptosis in colorectal cancer cells.

Long noncoding RNAs (lncRNAs) are emerging as having multiple roles in cancer progression. However, roles of lncRNAs in chemotherapy for colorectal cancer (CRC) remain unclear. This study investigated the biological functions of lncRNA PANDAR in CRC cells treated with curcumin chemotherapy. Herein, we identified that PANDAR expression was not notably differential in CRC tissues compared with the corresponding normal tissues. Consistently, in vitro experiments revealed that knockdown of PANDAR could not change the proliferation, apoptosis, or senescence of CRC cells. Further analyses showed that low-dose curcumin could induce senescence in CRC cells without affecting cell apoptosis. Moreover, expression of PANDAR was increased in curcumin-treated CRC cells. Furthermore, silencing PANDAR in curcumin-treated cells increased apoptosis and greatly attenuated senescence possibly by stimulating the expression of PUMA. Together, these findings indicate that knockdown of lncRNA PANDAR switches curcumin-induced senescence to apoptosis, which may be potentially valuable in CRC therapy.

A-kinase anchor protein 4 (AKAP4) a promising therapeutic target of colorectal cancer.

BackgroundColorectal cancer (CRC) ranks third among the estimated cancer cases and cancer related mortalities in the Western world. Early detection and efficient therapy of CRC remains a major health challenge. Therefore, there is a need to identify novel tumor markers for early diagnosis and treatment of CRC.MethodsA-kinase anchor protein 4 (AKAP4) gene and protein expression was monitored by quantitative polymerase chain reaction (qPCR), reverse transcription (RT)-PCR and Western blotting in normal colon tissue lysate, normal colon epithelial cells and in colon cancer cell lines viz., Caco-2, COLO205, COLO320DM, HCT-15, HCT116, HT-29, SW480, and SW620. The effect of AKAP4 on cellular growth, migration and invasion abilities was studied using gene silencing approach. The role of AKAP4 in various pathways involved in cell cycle, apoptosis, senescence was investigated in in vitro and in human xenograft mouse model.ResultsOur studies showed that AKAP4 gene and protein expression was expressed in all colon cancer cells while no expression was detectable in normal colon cells. Ablation of AKAP4 led to reduced cellular growth, migration, invasion and increased apoptosis and senescence of CRC cells in in vitro assays and tumor growth in human xenograft mouse. Human colon xenograft studies showed a significant decrease in the levels of cyclins B1, D and E and cyclin dependent kinases such as CDK1, CDK2, CDK4 and CDK6. Interestingly, an up-regulation in the levels of p16 and p21 was also observed. Besides, an increase in the levels of pro-apoptotic molecules AIF, APAF1, BAD, BID, BAK, BAX, PARP1, NOXA, PUMA and cyt-C and Caspase 3, 7, 8 and 9 was also found in cancer cells as well as in xenograft tissue sections. However, anti-apoptotic molecules BCL2, Bcl-xL, cIAP2, XIAP, Axin2 and Survivin were down regulated in these samples. Our data also revealed elevated expression of epithelial marker E-cadherin and down regulation of EMT markers N-cadherin, P-cadherin, SLUG, α-SMA, SNAIL, TWIST and Vimentin. Further ablation of AKAP4 resulted in the down regulation of invasion molecules matrix metalloproteinase MMP2, MMP3 and MMP9.ConclusionAKAP4 appears to be a novel CRC-associated antigen with a potential for developing as a new clinical therapeutic target.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-015-0258-y) contains supplementary material, which is available to authorized users.

FL118 induces p53-dependent senescence in colorectal cancer cells by promoting degradation of MdmX.

Anticancer agent FL118 was recently identified in screening of small-molecule inhibitors of human survivin expression. Although FL118 is a camptothecin analogue, its antitumor potency is much superior to other FDA-approved camptothecin analogues (irinotecan and topotecan). The mechanism of action (MOA) underlying the antitumor effects of FL118 remains to be fully elucidated. Here, we report that FL118 activates tumor suppressor p53 as a novel MOA in p53 wild-type cancer cells. Our studies show that this MOA involves an induction of proteasomal degradation of MdmX, a critical negative regulator of p53, in a manner largely independent of ATM-dependent DNA damage signaling pathway but dependent on E3-competent Mdm2. FL118 inhibits p53 polyubiquitination and monoubiquitination by Mdm2-MdmX E3 complex in cells and in cell-free systems. In contrast, FL118 stimulates Mdm2-mediated MdmX ubiquitination. Coimmunoprecipitation revealed that FL118 slightly decreases Mdm2-p53 interactions and moderately increases Mdm2-MdmX interactions, suggesting a change of targeting specificity of Mdm2-MdmX E3 complex from p53 to MdmX, resulting in accelerated MdmX degradation. As a result, p53 ubiquitination by Mdm2-MdmX E3 complex is reduced, which in turn activates p53 signaling. Activation of the p53 pathway by FL118 induces p53-dependent senescence in colorectal cancer cells. However, in the absence of p53 or in the presence of MdmX overexpression, FL118 promotes p53-independent apoptosis. These two distinct cellular consequences collectively contribute to the potent effects of FL118 to inhibit clonogenic potential of colon cancer cells. This study identifies a potential application of FL118 as an MdmX inhibitor for targeted therapies.

β-Asarone induces senescence in colorectal cancer cells by inducing lamin B1 expression

Colorectal cancer is a leading cause of cancer mortality with a complex carcinogenesis that includes reduced cellular senescence. Lamin proteins are decreased in senescing cells, and frequently decreased in malignancies. This study identified a new drug candidate for colorectal cancer that appears to target cell senescence via a lamin protein. β-Asarone (1-propenyl-2,4,5-methoxybenzol) is a compound from the traditional medical herb Acorus calamus Linn. This study tested the in vitro and in vivo effects of β-asarone on colorectal cancer cells by testing cell viability using human colorectal cell lines HT29 and SW480 in MTT assays; tumorigenesis using xenografts in nude mice and a mouse model of colorectal cancer; cell senescence using senescence-associated β-galactosidase activity; and expression of cancer and senescence-related proteins, specifically lamins, Oct-1, p53, p21, and p15, by Western blot. β-Asarone appeared to increase expression of lamin B1, p53, p21, but not lamin A/C. β-Asarone regulates p15 expression by regulation of Oct-1 binding. Collectively, the results suggested that β-asarone inhibits colon cancer formation in vivo and in vitro by inducing senescence. Since β-asarone induced lamin B1 expression, a model is proposed in which β-asarone inhibits colorectal cancer by inducing senescence through lamin B1.

Oridonin induces apoptosis and senescence by increasing hydrogen peroxide and glutathione depletion in colorectal cancer cells

We recently demonstrated that oridonin could induce apoptosis and senescence of colon cancer cells in vitro and in vivo. However, the underlying mechanism remains unknown. In this study, the involvement of reactive oxygen species in oridonin-induced cell death and senescence was investigated in colon adenocarcinoma-derived SW1116 cells. Oridonin increased intracellular hydrogen peroxide levels and reduced the glutathione content in a dose-dependent manner. N-acetylcysteine, a reactive oxygen species scavenger, not only blocked the oridonin-induced increase in hydrogen peroxide and glutathione depletion, but also blocked apoptosis and senescence induced by oridonin, as evidenced by the decrease in Annexin V and senescence-associated β-galactosidase- positive cells and the inhibition of oridonin-induced upregulation of p53 and p16 and downregulation of c-Myc. Moreover, exogenous catalase could inhibit the increase in hydrogen peroxide and apoptosis induced by oridonin, but not the glutathione depletion and senescence. Furthermore, thioredoxin reductase (TrxR) activity was reduced by oridonin in vitro and in cells, which may cause the increase in hydrogen peroxide. In conclusion, the increase in hydrogen peroxide and glutathione depletion account for oridonin-induced apoptosis and senescence in colorectal cancer cells, and TrxR inhibition is involved in this process. Given the importance of TrxR as a novel cancer target in colon cancer, oridonin would be a promising clinical candidate. The mechanism of oridonin-induced inhibition of TrxR warrants further investigation.

Inhibition of VEGF induces cellular senescence in colorectal cancer cells

Vascular endothelial growth factor (VEGF) inhibitors, such as bevacizumab, have improved outcomes in metastatic colorectal cancer (CRC). Recent studies have suggested that VEGF can delay the onset of cellular senescence in human endothelial cells. As VEGF receptors are known to be upregulated in CRC, we hypothesized that VEGF inhibition may directly influence cellular senescence in this disease. In our study, we observed that treatment with bevacizumab caused a significant increase (p < 0.05) in cellular senescence in vitro in several CRC cells, such as MIP101, RKO, SW620 and SW480 cells, compared to untreated or human IgG-treated control cells. Similar results were also obtained from cells treated with a VEGFR2 kinase inhibitor Ki8751. In vivo, cellular senescence was detected in MIP101 tumor xenografts from 75% of mice treated with bevacizumab, while cellular senescence was undetectable in xenografts from mice treated with saline or human IgG (p < 0.05). Interestingly, we also observed that the proportion of senescent cells in colon cancer tissues obtained from patients treated with bevacizumab was 4.4-fold higher (p < 0.01) than those of untreated patients. To understand how VEGF inhibitors may regulate cellular senescence, we noted that among the two important regulators of senescent growth arrest of tumor cells, bevacizumab-associated increase in cellular senescence coincided with an upregulation of p16 but appeared to be independent of p53. siRNA silencing of p16 gene in MIP101 cells suppressed bevacizumab-induced cellular senescence, while silencing of p53 had no effect. These findings demonstrate a novel antitumor activity of VEGF inhibitors in CRC, involving p16.