PRDX2 promotes the proliferation of colorectal cancer cells by increasing the ubiquitinated degradation of p53

Colorectal cancer is the most common gastrointestinal cancer and causes severe damage to human health. PRDX2 is a member of the peroxiredoxin family reported to have a high level of expression in colorectal cancer. However, the mechanisms by which PRDX2 promotes the proliferation of colorectal cancer are still unclear. Here, the results indicated that PRDX2 expression was upregulated in colorectal cancer and closely correlated with poor prognosis. Functionally, PRDX2 promoted the proliferation of colorectal cancer cells. Mechanistically, PRDX2 could bind RPL4, reducing the interaction between RPL4 and MDM2. These findings demonstrate that the oncogenic property of PRDX2 may be attributed to its regulation of the RPL4-MDM2-p53 pathway, leading to p53 ubiquitinated degradation.Subject terms: Colorectal cancer, Colorectal cancer     

Autophagy regulates apoptosis by targeting NOXA for degradation

Apoptosis and autophagy mutually regulate various cellular physiological and pathological processes. The crosstalk between autophagy and apoptosis is multifaceted and complicated. Elucidating the molecular mechanism of their crosstalk will advance the therapeutic applications of autophagy for treating cancer and other diseases. NOXA, a BH3-only member of the BCL-2 family, was reported to induce apoptosis and promote autophagy. Here, we report that autophagy regulates apoptosis by targeting NOXA for degradation. Inhibiting autophagy increases NOXA protein levels by extending the protein half-life. NOXA accumulation effectively suppresses tumor cell growth by inducing apoptosis, which is further enhanced when p53 is present. Mechanistically, NOXA is hijacked by p62 as autophagic cargo, and its three lysine residues at the C-terminus are necessary for NOXA degradation in lysosomes. Taken together, our study demonstrates that NOXA serves as a bridge in the crosstalk between autophagy and apoptosis and implies that autophagy inhibitors could be an effective therapy for cancer, especially wild-type p53-containing cancer.     

Inhibition of KHSRP sensitizes colorectal cancer to 5-fluoruracil through miR-501-5p-mediated ERRFI1 mRNA degradation

K-homology (KH)-type splicing regulatory protein (KHSRP) is an RNA binding protein that participates in RNA variable splicing and stability, and facilitates the biogenesis of miRNAs that target mRNA. However, to date, the role of KHSRP in colorectal cancer (CRC) progression has not been reported. In this study, the function of KHSRP in CRC proliferation and 5-fluoruracil (5-FU) resistance was investigated. The upregulation of KHSRP expression was confirmed in CRC patient tissues and two CRC cell lines. Manipulating KHSRP expression altered cell proliferation and 5-FU resistance in CRC cells. ERRFI1, a downstream effector of KHSRP in CRC cells, reduced CRC cell proliferation. Sensitivity to 5-FU mediated by KHSRP knockdown was reversed by ERRFI1 knockdown. We found that KHSRP decreased ERRFI1 mRNA expression indirectly. By screening KHSRP-regulated miRNAs, we further found that miR-501-5p directly combines with KHSRP in CRC cells. Mechanistically, the results of a luciferase assay suggested that miR-501-5p directly binds to the ERRFI1 3′-untranslated region. Taken together, our data indicated that modification of ERRFI1 by KHSRP occurs through miR-501-5p, an essential mechanism driving CRC proliferation and 5-FU resistance. Insight into this mechanism may provide novel targets for overcoming drug resistance in CRC.     

Regulation of PRDX1 peroxidase activity by Pin1

Pin1 isomerizes the phosphorylated Ser/Thr-Pro peptide bonds and regulates the functions of its binding proteins by inducing conformational changes. Involvement of Pin1 in the aging process has been suggested based on the phenotype of Pin1-knockout mice and its interaction with lifespan regulator protein, p66^Shc. In this study, we utilize a proteomic approach and identify peroxiredoxin 1 (PRDX1), another regulator of aging, as a novel Pin1 binding protein. Pin1 binds to PRDX1 through interacting with the phospho-Thr⁹⁰-Pro⁹¹ motif of PRDX1, and this interaction is abolished when the Thr⁹⁰ of PRDX1 is mutated. The Pin1 binding motif, Thr-Pro, is conserved in the 2-Cys PRDXs, PRDX1–4 and the interactions between Pin1 and PRDX2–4 are also demonstrated. An increase in hydrogen peroxide buildup and a decrease in the peroxidase activity of 2-Cys PRDXs were observed in Pin1^?/? mouse embryonic fibroblasts (MEFs), with the activity of PRDXs restored when Pin1 was re-introduced into the cells. Phosphorylation of PRDX1 at Thr⁹⁰ has been shown to inhibit its peroxidase activity; however, how exactly the activity of PRDX1 is regulated by phosphorylation still remains unknown. Here, we demonstrate that Pin1 facilitates the protein phosphatase 2A-mediated dephosphorylation of PRDX1, which helps to explain the accumulation of the inactive phosphorylated form of PRDX1 in Pin1^?/? MEFs. Collectively, we identify Pin1 as a novel PRDX1 binding protein and propose a mechanism for Pin1 in regulating the metabolism of reactive oxygen species in cells.     

SSRP1 promotes colorectal cancer progression and is negatively regulated by miR‐28‐5p

In this study, microarray data analysis, real‐time quantitative PCR and immunohistochemistry were used to detect the expression levels of SSRP1 in colorectal cancer (CRC) tissue and in corresponding normal tissue. The association between structure‐specific recognition protein 1 (SSRP1) expression and patient prognosis was examined by Kaplan‐Meier analysis. SSRP1 was knocked down and overexpressed in CRC cell lines, and its effects on proliferation, cell cycling, migration, invasion, cellular energy metabolism, apoptosis, chemotherapeutic drug sensitivity and cell phenotype‐related molecules were assessed. The growth of xenograft tumours in nude mice was also assessed. MiRNAs that potentially targeted SSRP1 were determined by bioinformatic analysis, Western blotting and luciferase reporter assays. We showed that SSRP1 mRNA levels were significantly increased in CRC tissue. We also confirmed that this upregulation was related to the terminal tumour stage in CRC patients, and high expression levels of SSRP1 predicted shorter disease‐free survival and faster relapse. We also found that SSRP1 modulated proliferation, metastasis, cellular energy metabolism and the epithelial‐mesenchymal transition in CRC. Furthermore, SSRP1 induced apoptosis and SSRP1 knockdown augmented the sensitivity of CRC cells to 5‐fluorouracil and cisplatin. Moreover, we explored the molecular mechanisms accounting for the dysregulation of SSRP1 in CRC and identified microRNA‐28‐5p (miR‐28‐5p) as a direct upstream regulator of SSRP1. We concluded that SSRP1 promotes CRC progression and is negatively regulated by miR‐28‐5p.     

LAMTOR5 expression level is a biomarker for colorectal cancer and lncRNA LAMTOR5-AS1 predicting miRNA sponging effect

Molecular Biology Reports - Strong evidence indicated that high expression of HBXIP (also known as LAMTOR5) promotes cancer cells proliferation and helps cancer progression. Long non-coding RNAs...     

LIN28B/IRS1 axis is targeted by miR-30a-5p and promotes tumor growth in colorectal cancer

Insulin receptor substrate 1 (IRS1) is a potential oncogene that has been implicated in several malignant tumors. However, the regulatory mechanism of IRS1 remains to be investigated. The aim of our current study is to unveil the mechanism by which IRS1 exerts functions in tumorigenesis of colorectal cancer (CRC). The expression level of IRS1 was found to be higher in CRC cells in comparison with the normal cell. To determine the role of IRS1 in regulating CRC cellular processes, loss-of-function assays were designed and carried out in two CRC cell lines. Both in vitro and in vivo functional assays indicated that silencing of IRS1 suppressed CRC cell survival. Based on bioinformatics prediction and mechanism experiments, IRS1 was identified as a downstream target of miR-30a-5p. Furthermore, RNA-binding protein lin-28 homolog B (LIN28B) was determined to be a stabilizer of IRS1 messenger RNA. More importantly, LIN28B also acted as a target of miR-30a-5p.Through rescue assays, we proved that LIN28B-stablized IRS1 mediated miR-30a-5p-mediated CRC cell growth. In conclusion, this study revealed that LIN28B and LIN28B-stablized IRS1 promoted CRC cell growth by cooperating with miR-30a-5p.     

NOXA, a sensor of proteasome integrity, is degraded by 26S proteasomes by an ubiquitin-independent pathway that is blocked by MCL-1

Ubiquitin (Ub)-mediated proteasome-dependent proteolysis is critical in regulating multiple biological processes including apoptosis. We show that the unstructured BH3-only protein, NOXA, is degraded by an Ub-independent mechanism requiring 19S regulatory particle (RP) subunits of the 26S proteasome, highlighting the possibility that other unstructured proteins reported to be degraded by 20S proteasomes in vitro may be bona fide 26S proteasome substrates in vivo. A lysine-less NOXA (NOXA-LL) mutant, which is not ubiquitinated, is degraded at a similar rate to wild-type NOXA. Myeloid cell leukemia 1, but not other anti-apoptotic BCL-2 family proteins, stabilizes NOXA by interaction with the NOXA BH3 domain. Depletion of 19S RP subunits, but not alternate proteasome activator REG subunits, increases NOXA half-life in vivo. A NOXA-LL mutant, which is not ubiquitinated, also requires an intact 26S proteasome for degradation. Depletion of the 19S non-ATPase subunit, PSMD1 induces NOXA-dependent apoptosis. Thus, disruption of 26S proteasome function by various mechanisms triggers the rapid accumulation of NOXA and subsequent cell death strongly implicating NOXA as a sensor of 26S proteasome integrity.     

Proteomic expression analysis of surgical human colorectal cancer tissues: up-regulation of PSB7, PRDX1, and SRP9 and hypoxic adaptation in cancer

Colorectal adenocarcinoma is one of the worldwide leading causes of cancer deaths. Discovery of specific biomarkers for early detection of cancer progression and the identification of underlying pathogenetic mechanisms are important tasks. Global proteomic approaches have thus far been limited by the large dynamic range of molecule concentrations in tissues and the lack of selective enrichment of the low-abundance proteome. We studied paired cancerous and normal clinical tissue specimens from patients with colorectal adenocarcinomas by heparin affinity fractionation enrichment (HAFE) followed by 2-D PAGE and tandem mass spectrometric (MS/MS) identification. Fifty-six proteins were found to be differentially expressed, of which 32 low-abundance proteins were only detectable after heparin affinity enrichment. MS/MS was used to identify 5 selected differentially expressed proteins as proteasome subunit beta type 7 (PSB7), hemoglobin alpha subunit (HBA), peroxiredoxin-1 (PRDX1), argininosuccinate synthase (ASSY), and signal recognition particle 9 kDa protein (SRP9). This is the first proteomic study detecting the differential expression of these proteins in human colorectal cancer tissue. Several of the proteins are functionally related to tissue hypoxia and hypoxic adaptation. The relative specificities of PSB7, PRDX1, and SRP9 overexpression in colon cancer were investigated by Western blot analysis of patients with colon adenocarcinomas and comparison with a control cohort of patients with lung adenocarcinomas. Furthermore, immunohistochemistry on tissue sections was used to define the specific locations of PSB7, PRDX1, and SRP9 up-regulation within heterogeneous primary human tumor tissue. Overexpression of the three proteins was restricted to the neoplastic cancer cell population within the tumors, demonstrating both cytoplasmic and nuclear localization of PSB7 and predominantly cytoplasmic localization of PRDX1 and SRP9. In summary, we describe heparin affinity fractionation enrichment (HAFE) as a prefractionation tool for the study of the human primary tissue proteome and the discovery of PSB7, PRDX1, and SRP9 up-regulation as candidate biomarkers of colon cancer.     

miR-548c-5p inhibits colorectal cancer cell proliferation by targeting PGK1

Accumulating studies have implicated that microRNAs (miRNAs) are involved in the pathogenesis of colorectal cancer (CRC). However, the role of miR-548c-5p, a novel identified miRNA in malignancies, in colorectal carcinogenesis remains largely unknown. The present study is aimed to investigate the effect and molecular mechanism of miR-548c-5p in CRC by a sequence of cellular experiments. miR-548c-5p was significantly downregulated, whereas phosphoglycerate kinase 1 (PGK1), a key enzyme for glycolysis, was obviously upregulated in peripheral blood mononuclear cells and cancer tissues from patients with CRC. Besides, miR-548c-5p and PGK1 were negatively associated with each other. The luciferase reporter assay revealed that PGK1 was a targeted gene of miR-548c-5p. Moreover, the proliferation and generation of inflammatory cytokines (TNF-α and IL-6) were significantly inhibited in miR-548c-5p-overexpressed SW480 CRC cells stimulated by lipopolysaccharide (LPS). Accordingly, miR-548c-5p may serve as a cancer suppressor in CRC by targeting PGK1.     

25-Hydroxyvitamin D-1alpha-hydroxylase activity is diminished in human prostate cancer cells and is enhanced by gene transfer

The hormone 1alpha,25-dihydroxyvitamin D (1alpha,25(OH)(2)D) inhibits growth and induces differentiation of prostate cells. The enzyme responsible for 1alpha,25(OH)(2)D synthesis, 25-hydroxyvitamin D (25(OH)D)-1alpha-hydroxylase (1alpha-OHase), has been demonstrated in human prostate cells. We compared the levels of 1alpha-OHase activity in prostate cancer cell lines, LNCaP, DU145 and PC-3 and in primary cultures of normal, cancerous and benign prostatic hyperplasia (BPH) prostate cells. We observed a marked decrease in 1alpha-OHase activity in prostate cancer cells, including an undetectable level of activity in LNCaP cells. Transient or stable transfection of 1alpha-OHase cDNA into LNCaP cells increased 1alpha-OHase activity from undetectable to 4.95pmole/mg+/-0.69pmole/mg and 5.8pmole/mg+/-0.7pmole/mg protein per hour, respectively. In response to 25(OH)D, the prohormone of 1alpha,25(OH)(2)D, the transfected LNCaP cells showed a significant inhibition of 3H-thymidine incorporation (37%+/-6% and 56%+/-4% at 10(-8)M for transiently and stably transfected cells, respectively). These findings support an important autocrine role for 1alpha,25(OH)(2)D in the prostate and suggest that the re-introduction of the 1alpha-OHase gene to prostate cancer cells, in conjunction with the systemic administration of 25(OH)D, constitutes an endocrine form of gene therapy that may be less toxic than the systemic administration of 1alpha,25(OH)(2)D.     

The formation of highly soluble oligomers of alpha-synuclein is regulated by fatty acids and enhanced in Parkinson's disease

Accumulation of misfolded proteins as insoluble aggregates occurs in several neurodegenerative diseases. In Parkinson's disease (PD) and dementia with Lewy bodies (DLB), alpha-synuclein (alpha S) accumulates in insoluble inclusions. To identify soluble alpha S oligomers that precede insoluble aggregates, we probed the cytosols of mesencephalic neuronal (MES) cells, normal and alpha S-transgenic mouse brains, and normal, PD, and DLB human brains. All contained highly soluble oligomers of alpha S whose detection was enhanced by delipidation. Exposure of living MES neurons to polyunsaturated fatty acids (PUFAs) increased alpha S oligomer levels, whereas saturated FAs decreased them. PUFAs directly promoted oligomerization of recombinant alphaS. Transgenic mice accumulated soluble oligomers with age. PD and DLB brains had elevated amounts of the soluble, lipid-dependent oligomers. We conclude that alpha S interacts with PUFAs in vivo to promote the formation of highly soluble oligomers that precede the insoluble alpha S aggregates associated with neurodegeneration.     

HERC3 regulates epithelial-mesenchymal transition by directly ubiquitination degradation EIF5A2 and inhibits metastasis of colorectal cancer

E3 ligase is widely reported to exert fundamental functions in cancers. Through rigorous bioinformatic analysis concentrating E3 ligases based on data from Genotype-Tissue Expression (GTEx) and data from The Cancer Genome Atlas (TCGA), HERC3 was indicated to be downregulated in colorectal cancer (CRC) and HERC3 downregulation showed poor overall survival (OS) and disease-free survival (DFS). Through qRT-PCR, western blotting and Immunohistochemistry (IHC), analytical results were validated based on tissues in Zhongshan hospital. Functionally, HERC3 was indicated to inhibit the migration, invasion and metastasis in vitro and in vivo through transwell assays, wound healing assays and vivo experiments. And HERC3 could regulate epithelial-mesenchymal transition (EMT) in CRC. Furthermore, immunoprecipitation (IP), coimmunoprecipitation (co-IP) and GST-pulldown assays indicated that HERC3 could directly interact with EIF5A2 in vitro and in vivo through the RCC1 domain in HERC3. And HERC3 could function as an E3 to promote the K27 and K48-linked ubiquitination degradation of EIF5A2 via the HECT domain in HERC3, besides, K47, K67, K85, and K121 in EIF5A2 were identified as ubiquitination sites. In addition, HERC3 was indicated to affect the migration, invasion and metastasis and further regulatE EMT via EIF5A2/TGF-/Smad2/3 signal. The present study may provide insight into the mechanism of EMT in CRC.Subject terms: Ubiquitins, Gastrointestinal cancer     

FZD6 expression is negatively regulated by miR-199a-5p in human colorectal cancer

Colorectal cancer (CRC), the third most common cancer worldwide, also has the highest rate of cancer-related morbidity and mortality. WNT signaling is initiated by binding of WNT to various receptors, including frizzleds (FZDs), and plays a critical role in CRC and other tumor development by regulating proliferation, differentiation, migration, apoptosis, and polarity. Among the members of the FZD family, FZD6 is broadly expressed in various tissues, and its overexpression has been reported in several cancers, suggesting an important role in cancer development. In this study, we investigated the expression of FZD6 in patients with CRC and found it to be increased in tumors, as compared to paired adjacent non-tumor tissues. Additionally, we found that FZD6 expression was negatively regulated by miR199a5p in CRC cells. These results suggest that overexpression of FZD6, mediated by reduced expression of miR-199a-5p, may play an important role in the development of CRC. [BMB Reports 2015; 48(6): 360-366]     

Geldanamycin-induced degradation of Chk1 is mediated by proteasome

Checkpoint kinase 1 (Chk1) is a cell cycle regulator and a heat shock protein 90 (Hsp90) client. It is essential for cell proliferation and survival. In this report, we analyzed the mechanisms of Chk1 regulation in U87MG glioblastoma cells using Geldanamycin (GA), which interferes with the function of Hsp90. GA reduced Chk1 protein level but not its mRNA level in glioblastoma cells. Co-treatment with GA and cycloheximide (CHX), a protein synthesis inhibitor, induced a decrease of half-life of the Chk1 protein to 3h and resulted in Chk1 down-regulation. CHX alone induced only 32% reduction of Chk1 protein even after 24h. These findings indicated that reduction of Chk1 by GA was due to destabilization and degradation of the protein. In addition, GA-induced down-regulation of Chk1 was reversed by MG132, a specific proteasome inhibitor. And it was revealed that Chk1 was ubiquitinated by GA. These results have indicated that degradation of Chk1 by GA was mediated by the ubiquitin-proteasome pathway in U87MG glioblastoma cells.     

Inhibition of proteasomal degradation of Mcl-1 by cobalt chloride suppresses cobalt chloride-induced apoptosis in HCT116 colorectal cancer cells

Cobalt promotes apoptosis in multiple cell systems, however, the molecular mechanisms that influence cobalt-induced apoptosis are not fully understood. We investigated mechanisms of cobalt chloride induced apoptosis in HCT116 colorectal cancer cells. Cobalt chloride induced dose dependent apoptosis in HCT116 cells (250–750 μM) which, at higher concentrations (500–750 μM), was associated with an increase in the expression of the Bcl-2-related Mcl-1 survival protein. Cobalt chloride caused the accumulation of higher molecular weight ubiquitin-conjugates of Mcl-1 in intact HCT116 cells and inhibited the activity of the trypsin-like site of the 20S proteasome in an in vitro assay. Although siRNA-mediated knockdown of Mcl-1 increased apoptosis in HCT116 cells, the combination of Mcl-1 siRNA and cobalt chloride induced very high levels of cell killing. Therefore, inhibition of the proteasome by cobalt chloride leads to the accumulation of Mcl-1 which acts to limit cobalt chloride induced apoptosis.