LKB1 loss promotes colorectal cancer cell metastasis through regulating TNIK expression and actin cytoskeleton remodeling.

Colorectal cancer (CRC) is one of the most common malignant tumors. Approximately 5%-6% of CRC cases are associated with hereditary CRC syndromes, including the Peutz-Jeghers syndrome (PJS). Liver kinase B1 (LKB1), also known as STK11, is the major gene responsible for PJS. LKB1 heterozygotic deficiency is involved in intestinal polyps in mice, while the mechanism of LKB1 in CRC remains elusive. In this study, we generated LKB1 knockout (KO) CRC cell lines by using CRISPR-Cas9. LKB1 KO promoted CRC cell motility in vitro and tumor metastases in vivo. LKB1 attenuated expression of TRAF2 and NCK-interacting protein kinase (TNIK) as accessed by RNA-seq and western blots, and similar suppression was also detected in the tumor tissues of azoxymethane/dextran sodium sulfate-induced intestinal-specific LKB1-KO mice. LKB1 repressed TNIK expression through its kinase activity. Moreover, attenuating TNIK by shRNA inhibited cell migration and invasion of CRC cells. LKB1 loss-induced high metastatic potential of CRC cells was depended on TNIK upregulation. Furthermore, TNIK interacted with ARHGAP29 and further affected actin cytoskeleton remodeling. Taken together, LKB1 deficiency promoted CRC cell metastasis via TNIK upregulation and subsequently mediated cytoskeleton remodeling. These results suggest that LKB1-TNIK axis may play a crucial role in CRC progression.

Celastrol acts as a new histone deacetylase inhibitor to inhibit colorectal cancer cell growth via regulating macrophage polarity.

The tumorigenesis and progression of colorectal cancer are closely related to the tumor microenvironment, especially inflammatory response. Inhibitors of histone deacetylase (HDAC) have been reported as epigenetic regulators of the immune system to treat cancer and inflammatory diseases and our results demonstrated that Celastrol could act as a new HDAC inhibitor. Considering macrophages as important members of the tumor microenvironment, we further found that Celastrol could influence the polarization of macrophages to inhibit colorectal cancer cell growth. Specially, we used the supernatant of HCT116 and SW480 cells to induce Ana-1 cells in vitro and chose the spontaneous colorectal cancer model APCmin/+ mice as an animal model to validate in vivo. The results indicated that Celastrol could reverse the polarization of macrophages from M2 to M1 through impacting the colorectal tumor microenvironment both in vitro and in vivo. Furthermore, using bioinformatics analysis, we found that Celastrol might mechanistically polarize the macrophages through MAPK signaling pathway. In conclusion, our findings identified that Celastrol as a new HDAC inhibitor and suggested that Celastrol could modulate macrophage polarization, thus inhibiting colorectal cancer growth, which may provide some novel therapeutic strategies for colorectal cancer.

Smoking-related cancer death among men and women in an ageing society (China 2020-2040): a population-based modelling study.

BACKGROUND: China is experiencing a postpeak smoking epidemic with accelerating population ageing. Understanding the impacts of these factors on the future cancer burden has widespread implications. METHODS: We developed predictive models to estimate smoking-related cancer deaths among men and women aged ≥35 years in China during 2020-2040. Data sources for model parameters included the United Nations World Population Prospects, China Death Surveillance Database, national adult tobacco surveys and the largest national survey of smoking and all causes of death to date. The main assumptions included stable sex-specific and age-specific cancer mortality rates and carcinogenic risks of smoking over time. RESULTS: In a base-case scenario of continuing trends in current smoking prevalence (men: 57.4%-50.5%; women: 2.6%-2.1% during 2002-2018), the smoking-related cancer mortality rate with population ageing during 2020-2040 would rise by 44.0% (from 337.2/100 000 to 485.6/100 000) among men and 52.8% (from 157.3/100 000 to 240.4/100 000) among women; over 20 years, there would be 8.6 million excess deaths (0.5 million more considering former smoking), and a total of 117.3 million smoking-attributable years of life lost (110.3 million (94.0%) in men; 54.1 million (46.1%) in working-age (35-64 years) adults). An inflection point may occur in 2030 if smoking prevalence were reduced to 20% (Healthy China 2030 goal), and 1.4 million deaths would be averted relative to the base-case scenario if the trend were maintained through 2040. CONCLUSIONS: Coordinated efforts are urgently needed to curtail a rising tide of cancer deaths in China, with intensified tobacco control being key.

SOX chemotherapy with anti-PD-1 and iNKT cell immunotherapies for stage IV gastric adenocarcinoma with liver metastases: A case report.

Gastric cancer (GC) is the fourth most common cancer worldwide, with overall 5-year survival rate of approximate 20%. Although multimodal treatments that combine surgery with chemotherapy and immunotherapy have been shown to improve survival, pathological complete response (pCR) is rare in advanced GC patients with liver metastases. Pre-clinical studies and clinical trials have demonstrated the antitumor efficacy of invariant natural killer T (iNKT) cells in various malignancies, including GC. While multimodal therapy comprised of chemotherapy, anti-programmed cell death-1 (PD-1) therapy, and iNKT cell immunotherapy have not been reported in GC patients. This case report describes the treatment of an early 60s patient diagnosed with advanced stage IVB (T1N1M1) adenocarcinomas of gastric cardia with liver metastases who received multimodal therapy comprised of SOX chemotherapy, anti-programmed cell death-1 (PD-1) therapy, and iNKT cell immunotherapy followed by surgical resection. Dramatic decreases in tumor area were observed in both the primary tumor and metastatic lesions following six cycles of SOX chemotherapy and iNKT cell immunotherapy, and four cycles of anti-PD-1 therapy. This combined treatment resulted in the transformation of a remarkably large, unresectable liver metastases into a resectable tumor, and the patient received total gastrectomy with D2 lymph node dissection and liver metastasectomy. Subsequent pathological examination detected no cancer cells in either the primary site or liver metastatic lesions, supporting the likelihood that this treatment achieved pCR. To our knowledge, this report represents the first case of a metastatic gastric cancer patient displaying pCR after six months of multimodal therapy, thus supporting that a SOX chemotherapy, anti-PD-1 therapy, and iNKT cell immunotherapy combination strategy may be effective for treating, and potentially curing, patients with advanced gastric adenocarcinoma.

Age-related macrophage alterations are associated with carcinogenesis of colorectal cancer.

Older age is a major risk factor for colorectal cancer. Macrophage is one of the most abundant immune cell types infiltrated in colorectal cancer, but the contribution of macrophages in elder tumor microenvironment is far from clear. In this study, we first detected the expression of CD206, CD68 in colorectal cancer tissues by multiplex fluorescence immunohistochemical staining. The infiltration of CD68+/CD206+ cells in tumor tissues from old patients was higher than those from young patients. When mixed with CT26 cells, both young and aged TAMs enhanced tumor growth of CT26 cells, but CT26 mixed with aged TAMs form larger tumors compared with young TAMs. CT26 formed more and larger tumors in the abdominal cavity of aged mice compared with young. Total macrophage infiltration and the CD206+ macrophages infiltration were both higher in aged mice compared with young mice. The expression signatures of tumor-associated macrophages altered with ageing and p-NF-κB translocation to nucleus was more significant in TAMs from aged mice compared with young. Our results showed that infiltration of macrophages in colorectal cancer tissues increased with ageing. Macrophages from aged host were more likely to polarize to pro-tumor phenotype, and more powerful in promoting tumor cell proliferation.

Mutation or not, what directly establishes a neoplastic state, namely cellular immortality and autonomy, still remains unknown and should be prioritized in our research.

Although the concept that cancer is caused by mutations has been widely accepted, there still are ample data deprecating it. For example, embryonic cells displaced in non-embryonic environments may develop to cancer, whereas cancer cells placed in embryonic environments may be reverted to phenotypic normal. Although many intracellular or extracellular aberrations are known to be able to initiate a lengthy tumorigenesis, the molecular or cellular alterations that directly establish a neoplastic state, namely cellular immortality and autonomy, still remain unknown. Hereditary traits are encoded not only by gene sequences but also by karyotype and DNA or chromosomal structures that may be altered via non-mutational mechanisms, such as post-translational modifications of nuclear proteins, to initiate tumorigenesis. However, the immortal and autonomous nature of neoplasms makes them "new" organisms, meaning that neoplasms should have mutations to distinguish themselves from their host patients in the genome. Neoplasms are malignant if they bear epigenetic or genetic alterations in mutator genes, i.e. the genes whose alterations accelerate other genes to mutate, whereas neoplasms are benign if their epigenetic or genetic aberrations occur only in non-mutator genes. Future mechanistic research should be focused on identifying the alterations that directly establish cellular immortality and autonomy. Benign tumors may have many fewer alterations and thus be much better models than cancers for such research. Future translational research should be aimed at identifying the cellular factors that control cancer cells' phenotypes and at establishing approaches of directing cancer cells towards differentiation, which should be a promising therapeutic tactic.

Upregulation of NOD1 and NOD2 contribute to cancer progression through the positive regulation of tumorigenicity and metastasis in human squamous cervical cancer.

BACKGROUND: Metastatic cervical squamous cell carcinoma (CSCC) has poor prognosis and is recalcitrant to the current treatment strategies, which warrants the necessity to identify novel prognostic markers and therapeutic targets. Given that CSCC is a virus-induced malignancy, we hypothesized that the pattern recognition receptors (PRRs) involved in the innate immune response likely play a critical role in tumor development. METHODS: A bioinformatics analysis, qPCR, IHC, immunofluorescence, and WB were performed to determine the expression of NOD1/NOD2. The biological characteristics of overexpression NOD1 or NOD2 CSCC cells were compared to parental cells: proliferation, migration/invasion and cytokines secretion were examined in vitro through CCK8/colony formation/cell cycle profiling/cell counting, wound healing/transwell, and ELISA assays, respectively. The proliferative and metastatic capacity of overexpression NOD1 or NOD2 CSCC cells were also evaluated in vivo. FCM, mRNA and protein arrays, ELISA, and WB were used to identify the mechanisms involved, while novel pharmacological treatment were evaluated in vitro and in vivo. Quantitative variables between two groups were compared by Student's t test (normal distribution) or Mann-Whitney U test (non-normal distribution), and one-way or two-way ANOVA was used for comparing multiple groups. Pearson χ2 test or Fisher's exact test was used to compare qualitative variables. Survival curves were plotted by the Kaplan-Meier method and compared by the log-rank test. P values of < 0.05 were considered statistically significant. RESULTS: NOD1 was highly expressed in CSCC with lymph-vascular space invasion (LVSI, P < 0.01) and lymph node metastasis (LM, P < 0.01) and related to worse overall survival (OS, P = 0.016). In vitro and in vivo functional assays revealed that the upregulation of NOD1 or NOD2 in CSCC cells promoted proliferation, invasion, and migration. Mechanistically, NOD1 and NOD2 exerted their oncogenic effects by activating NF-κb and ERK signaling pathways and enhancing IL-8 secretion. Inhibition of the IL-8 receptor partially abrogated the effects of NOD1/2 on CSCC cells. CONCLUSIONS: NOD1/2-NF-κb/ERK and IL-8 axis may be involved in the progression of CSCC; the NOD1 significantly enhanced the progression of proliferation and metastasis, which leads to a poor prognosis. Anti-IL-8 was identified as a potential therapeutic target for patients with NOD1high tumor.

XBP1 regulates the protumoral function of tumor-associated macrophages in human colorectal cancer.

Macrophages are among the most abundant immune cells in colorectal cancer (CRC). Re-educating tumor-associated macrophages (TAMs) to switch from protumoral to anti-tumoral activity is an attractive treatment strategy that warrants further investigation. However, little is known about the key pathway that is activated in TAMs. In this study, infitrating CD206+ TAMs in CRC were sorted and subjected to RNA-seq analysis. Differentially expressed genes were found to be enriched in unfolded protein response/endoplasmic reticulum stress response processes, and XBP1 splicing/activation was specifically observed in TAMs. XBP1 activation in TAMs promoted the growth and metastasis of CRC. Ablation of XBP1 inhibited the expression of the pro-tumor cytokine signature of TAMs, including IL-6, VEGFA, and IL-4. Simultaneously, XBP1 depletion could directly inhibit the expression of SIRPα and THBS1, thereby blocking "don't eat me" recognition signals and enhancing phagocytosis. Therapeutic XBP1 gene editing using AAV2-sgXBP1 enhanced the anti-tumor activity. Together, XBP1 activation in TAMs drives CRC progression by elevating pro-tumor cytokine expression and secretion, as well as inhibiting macrophage phagocytosis. Targeting XBP1 signaling in TAMs may be a potential strategy for CRC therapy.

Stimulation of hypothalamic oxytocin neurons suppresses colorectal cancer progression in mice.

Emerging evidence suggests that the nervous system is involved in tumor development in the periphery, however, the role of the central nervous system remains largely unknown. Here, by combining genetic, chemogenetic, pharmacological, and electrophysiological approaches, we show that hypothalamic oxytocin (Oxt)-producing neurons modulate colitis-associated cancer (CAC) progression in mice. Depletion or activation of Oxt neurons could augment or suppress CAC progression. Importantly, brain treatment with celastrol, a pentacyclic triterpenoid, excites Oxt neurons and inhibits CAC progression, and this anti-tumor effect was significantly attenuated in Oxt neuron-lesioned mice. Furthermore, brain treatment with celastrol suppresses sympathetic neuronal activity in the celiac-superior mesenteric ganglion (CG-SMG), and activation of ß2 adrenergic receptor abolishes the anti-tumor effect of Oxt neuron activation or centrally administered celastrol. Taken together, these findings demonstrate that hypothalamic Oxt neurons regulate CAC progression by modulating the neuronal activity in the CG-SMG. Stimulation of Oxt neurons using chemicals, for example, celastrol, might be a novel strategy for colorectal cancer treatment.

Colorectal (or 'bowel') cancer killed nearly a million people in 2018 alone: it is, in fact, the second leading cause of cancer death globally. Lifestyle factors and inflammatory bowel conditions such as chronic colitis can heighten the risk of developing the disease. However, research has also linked to the development of colorectal tumours to stress, anxiety and depression. This 'brain-gut' connection is particularly less-well understood. One brain region of interest is the hypothalamus, an almond-sized area which helps to regulate mood and bodily processes using chemical messengers that act on various cells in the body. For instance, Oxt neurons in the hypothalamus produce the hormone oxytocin which regulates emotional and social behaviours. These cells play an important role in modulating anxiety, stress and depression. To investigate whether they could also influence the growth of colorectal tumours, Pan et al. used various approaches to manipulate the activity of Oxt neurons in mice with colitis-associated cancer. Disrupting the Oxt neurons in these animals increased anxiety-like behaviours and promoted tumour growth. Stimulating these cells, on the other hand, suppressed cancer progression. Further experiments also showed that treating the mice with celastrol, a plant extract which can act on the hypothalamus, stimulated Oxt neurons and reduced tumour growth. In particular, the compound worked by acting on a nerve structure in the abdomen which relays messages to the gut. These preliminary findings suggest that the hypothalamus and its Oxt-producing neurons may influence the progression of colorectal cancer in mice by regulating the activity of an abdominal 'hub' of the nervous system. Modulating the activity of Oxt-producing neurons could therefore be a potential avenue for treatment.

EGFR-IL-6 Signaling Axis Mediated the Inhibitory Effect of Methylseleninic Acid on Esophageal Squamous Cell Carcinoma.

Epidemiological and experimental evidence indicate that selenium is associated with a reduced risk of some cancers, including esophageal cancer. However, the exact mechanism is still unclear. In the present study, we used esophageal squamous cell carcinoma (ESCC) cell lines and animal models to explore the anti-cancer mechanism of methylseleninic acid (MSA). Firstly, MSA treatment dramatically attenuated Epidermal Growth Factor Receptor (EGFR) protein expression but did not alter mRNA levels in ESCC cells. On the contrary, EGFR overexpression partly abolished the inhibitory effect of MSA. With a microRNA-array, we found MSA up-regulated miR-146a which directly targeted EGFR, whereas miR-146a inhibitor antagonized MSA-induced decrease of EGFR protein. We further used 4-nitroquinoline-1-oxide (4NQO)-induced esophageal tumor mice model to evaluate the inhibitory effect of MSA in vivo. MSA treatment significantly decreased the tumor burden and EGFR protein expression in tumor specimens. Furthermore, MSA treatment inhibited EGFR pathway and subsequntly reduced Interleukin-6 (IL-6) secretion in the supernatant of cancer cell lines. MSA-induced IL-6 suppression was EGFR-dependent. To further evaluate the association of IL-6 and the anti-tumor effect of MSA on esophageal cancer, we established the 4NQO-induced esophageal tumor model in IL-6 knock-out (IL-6 KO) mice. The results showed that IL-6 deficiency did not affect esophageal tumorigenesis in mice, but the inhibitory effect of MSA was abolished in IL-6 KO mice. In conclusion, our study demonstrated that MSA upregulated miR-146a which directly targeted EGFR, and inhibited EGFR protein expression and pathway activity, subsequently decreased IL-6 secretion. The inhibitory effect of MSA on esophageal cancer was IL-6 dependent. These results suggested that MSA may serve as a potential drug treating esophageal cancer.

Cetuximab enhances the anti-tumor function of macrophages in an IL-6 dependent manner.

AIMS: Cetuximab improves the survival of patients with advanced colorectal cancer (CRC). However, how cetuximab affects the tumor microenvironment has not been sufficiently understood. This study was to investigate whether cetuximab could inhibit the pro-tumor function of tumor-associated macrophages (TAMs) by suppressing the EGFR/IL-6 pathway. MAIN METHODS: The azoxymethane/dextran sodium sulfate (AOM/DSS) and tumor xenograft mouse models were used to assess the effect of cetuximab on TAMs. Flow cytometry, Western blotting, RT-qPCR, and ELISA were used to assess the prevalence of M2 and M1 phenotypes. Publicly available datasets of CRC patients were used to assess the relevance of EGFR and IL-6 expression as prognostic indicators. KEY FINDINGS: The two mouse models showed that cetuximab could attenuate the pro-tumor function of TAMs and decrease tumor burden. Cetuximab repolarized TAMs from M2-like to M1-like phenotypes, mainly by suppressing the IL-6 expression through NFκB and STAT3 pathways. Analysis of public scRNA-seq data indicated EGFR was mainly expressed on the surface of macrophage infiltration into tumor microenvironment. The public transcriptomics datasets showed that the expression level of IL-6 was positively correlated with EGFR in CRC patients, and PROGgeneV2 analysis indicated that IL-6 and CD206 both predicted poor recurrence-free and overall survival of CRC patients. Furthermore, the inhibition efficacy of cetuximab was significantly attenuated in IL-6 knockout CRC mice model. SIGNIFICANCE: These results indicate a new macrophage-based molecular mechanism explaining the effect of cetuximab in treatment of colorectal cancer.

EGFR targeting enhances the efficiency of chemotherapy through inhibiting IRE1α-XBP1s pathway in colorectal cancer cells.

Targeting EGFR combined with chemotherapy is one of the most valuable therapeutic strategies in colorectal cancer. However, resistance remains a major obstacle to improve efficacy. IRE1α-XBP1s signaling pathway is activated in many malignant tumors, and plays important roles in chemoresistance. Therefore, IRE1α-XBP1s might be a potential target to overcome the chemoresistance in colorectal cancer. In this study, we detected the activation of IRE1α-XBP1s signaling in patient cancer tissues and colorectal cancer cell lines. The phosphorylation level of IRE1α and the spliced XBP1s were aberrantly elevated in colorectal cancer, and IRE1α-XBP1s signaling activation was correlated with high EGFR expression. By overexpression of EGFR protein or activation by EGF treatment, we found that EGFR activation could enhance the phosphorylation of IRE1α and spliced XBP1s expression. On the contrary, inhibition of EGFR decreased the IRE1α-XBP1s signaling. Further, we examined the downstream signaling pathways regulated by EGFR. Inhibition of ERK activity could reverse the EGFR induced IRE1α-XBP1s activation. Co-IP confirmed the physical interaction of ERK and IRE1α. Cell growth and colony formation assay showed that the inhibition of IRE1α activity could suppress EGFR driven colorectal cancer cell proliferation. Furthermore, we found that oxaliplatin could activate IRE1α-XBP1s signaling, and combination with cetuximab partially reversed the activation. Inhibition of EGFR signaling could enhance the efficacy of oxaliplatin in vitro and in vivo. Our results showed that IRE1α RNase activity is aberrantly elevated in colorectal cancer, and EGFR signaling could activate IRE1α/XBP1s possibly through EGFR-MEK-ERK pathway. IRE1α-XBP1s pathway might involve in EGFR driven tumor cell proliferation. Cetuximab could partially recover oxaliplatin-induced IRE1α-XBP1s activation, and therefore enhance the anti-tumor efficacy of oxaliplatin. Our findings declare a new mechanism that targeting EGFR could inhibit chemotherapy-induced IRE1α-XBP1s activation and therefore enhance the efficacy.

Efficient isolation, culture, purification, and stem cell expression profiles of primary tumor cells derived from uterine cervical squamous cell carcinoma.

PROBLEM: Since not too many human uterus cervical squamous cell carcinoma (CSCC) cell lines in existence, efficient isolation, culture, and purification protocols for primary CSCC cells were optimized as a tool for the study of uterus CSCC. METHOD OF STUDY: The protocols for partial multiple enzymatic digestion and explant cell culture were combined and then the resulting mixed cell component cultures were purified by magnetic-activated cell sorting. Colony-forming assay was utilized for detection of cell carcinogenesis potential, and immunofluorescence was used to detect protein expression of CSCC. Finally, flow cytometry (FCM) was performed to analyze cancer stem cells (CSCs) phenotypic markers as well as programmed cell death ligand 1(PD-L 1). RESULTS: Freshly isolated cells containing tumor cells and cancer-associated fibroblasts (CAFs) efficiently proliferate to 85% confluence on a 6 cm petri dish in 5-7 days. Anti-epithelial cell adhesion molecule antibody (EpCAM) microbeads were used to successfully separate a homogeneous subpopulation of epithelial tumor cells. Both EpCAM+ and EpCAM- cell subpopulations were able to be passaged more than 30 times. Proportions of tumor cell populations expressed CSCs markers such as CD133, CD24, aldehyde dehydrogenase 1 (ALDH1), and CD44. The vimentin+ & EpCAM- population, defined with CAFs, could express CD146 mesenchymal stem cells marker. Meanwhile, PD-L 1 was identified in most subpopulation of CD44+ cells at low passage numbers. CONCLUSION: Efficient isolation, culture, and purification protocols for primary CSCC cells were successfully built. Additionally, the profiling of CSCs cell markers might provide promising therapeutic targets and clinic strategies.

Evidence for immortality and autonomy in animal cancer models is often not provided, which causes confusion on key issues of cancer biology.

Modern research into carcinogenesis has undergone three phases. Surgeons and pathologists started the first phase roughly 250 years ago, establishing morphological traits of tumors for pathologic diagnosis, and setting immortality and autonomy as indispensable criteria for neoplasms. A century ago, medical doctors, biologists and chemists started to enhance "experimental cancer research" by establishing many animal models of chemical-induced carcinogenesis for studies of cellular mechanisms. In this second phase, the two-hit theory and stepwise carcinogenesis of "initiation-promotion" or "initiation-promotion-progression" were established, with an illustrious finding that outgrowths induced in animals depend on the inducers, and thus are not authentically neoplastic, until late stages. The last 40 years are the third incarnation, molecular biologists have gradually dominated the carcinogenesis research fraternity and have established numerous genetically-modified animal models of carcinogenesis. However, evidence has not been provided for immortality and autonomy of the lesions from most of these models. Probably, many lesions had already been collected from animals for analyses of molecular mechanisms of "cancer" before the lesions became autonomous. We herein review the monumental work of many predecessors to reinforce that evidence for immortality and autonomy is essential for confirming a neoplastic nature. We extrapolate that immortality and autonomy are established early during sporadic human carcinogenesis, unlike the late establishment in most animal models. It is imperative to resume many forerunners' work by determining the genetic bases for initiation, promotion and progression, the genetic bases for immortality and autonomy, and which animal models are, in fact, good for identifying such genetic bases.

Clinical and functional significance of CHK1-S, an alternatively spliced isoform of the CHK1 gene, in hepatocellular carcinoma.

Alternative splicing plays critical roles in many disease processes and splicing dysregulation is a hallmark of cancer. The different splicing isoforms may have significantly different effects on the malignant progression of cancer. Checkpoint kinase 1 (CHK1) is a serine/threonine kinase and regulates DNA damage response. In this study, we measured the expression of an alternative CHK1 transcript (CHK1-S, excluded exon 3) in hepatocellular carcinoma (HCC) tissues. Our results showed that CHK1-S was significantly upregulated in HCC tissues compared with paired adjacent noncancerous hepatic tissues. The levels of full-length CHK1(CHK1-L), CHK1-S and the ratio of CHK1-S/L in tumor tissue were associated with relapse free survival (RFS) of postoperative HCC patients, respectively, but not the levels of CHK1-L, CHK1-S and the ratio of CHK1-S/L in adjacent normal tissue. To further demonstrate the role of CHK1-S in HCC, CCK-8 assays, EdU incorporation assays and colony formation assays were used. The results showed that overexpression of CHK1-S significantly accelerated HCC cell proliferation, compared with CHK1-L. In addition, we found that serine-arginine protein kinase 1 (SRPK1), as an upstream regulator kinase of splicing factor, could upregulate the expression of CHK1-S and its expression level was significantly higher in HCC tumors than the paired normal tissues and was associated with the levels of CHK1-S (P=0.016). In conclusion, our study demonstrated that CHK1-S, acts as an oncogene, which was upregulated and associated with RFS in HCC patients. SRPK1 may mediate its mRNA splicing in HCC. All these data indicated that the expression of CHK1-S would have potential prognostic values and splicing kinase SRPK1 might be developed as therapeutic target in HCC.

About three-fourths of mouse proteins unexpectedly appear at a low position of SDS-PAGE, often as additional isoforms, questioning whether all protein isoforms have been eliminated in gene-knockout cells or organisms.

Most genes in evolutionarily complex genomes are expressed to multiple protein isoforms, but there is not yet any simple high-throughput approach to identify these isoforms. Using an oversimplified top-down LC-MS/MS strategy, we detected, around the 26-kD position of SDS-PAGE, proteins produced from 782 genes in a Cdk4-/- mouse embryonic fibroblast cell line. Interestingly, only 213 (27.24%, about one-fourth) of these 782 genes have their proteins with a theoretical molecular mass (TMM) 10% smaller or larger than 26 kD, that is, between 23 and 29 kD, the range set as allowed variation in SDS-PAGE. These 213 proteins are considered as the wild type (WT). The remaining three-fourths includes proteins from 66 (9.44%) genes with a TMM smaller than 23 kD and proteins from 503 (64.32%, nearly two-thirds) genes with a TMM larger than 29 kD; these proteins are categorized into a larger-group or a smaller-group, respectively, for their appearance at a higher or lower position of SDS-PAGE. For instance, at this 26-kD position we detected proteins from the Rps27a, Snrpf, Hist1h4a, and Rps25 genes whose proteins' TMM is 8.6, 9.7, 11.4, and 13.7 kD, respectively, and detected proteins from the Plelc1 and Prkdc genes, whose largest isoform is 533.9 and 471.1 kD, respectively. We extrapolate that many of those proteins migrating unexpectedly in SDS-PAGE may be isoforms besides the WT protein. Moreover, we also detected a Cdk4 protein in this Cdk4-/- cell line, thus wondering whether some of other gene-knockout cells or organisms show similar incompleteness of the knockout.

SAE1 promotes human glioma progression through activating AKT SUMOylation-mediated signaling pathways.

BACKGROUND: The SUMO-activating enzyme SAE1 is indispensable for protein SUMOylation. A dysregulation of SAE1 expression involves in progression of several human cancers. However, its biological roles of SAE1 in glioma are unclear by now. METHODS: The differential proteome between human glioma tissues and para-cancerous brain tissues were identified by LC-MS/MS. SAE1 expression was further assessed by immunohistochemistry. The patient overall survival versus SAE1 expression level was evaluated by Kaplan-Meier method. The glioma cell growth and migration were evaluated under SAE1 overexpression or inhibition by the CCK8, transwell assay and wound healing analysis. The SUMO1 modified target proteins were enriched from total cellular or tissue proteins by incubation with the anti-SUMO1 antibody on protein-A beads overnight, then the SUMOylated proteins were detected by Western blot. Cell apoptosis and cell cycle were analyzed by flow cytometry. The nude mouse xenograft was determined glioma growth and tumorigenicity in vivo. RESULTS: SAE1 is identified to increase in glioma tissues by a quantitative proteomic dissection, and SAE1 upregulation indicates a high level of tumor malignancy grade and a poor overall survival for glioma patients. SAE1 overexpression induces an increase of the SUMOylation and Ser473 phosphorylation of AKT, which promotes glioma cell growth in vitro and in nude mouse tumor model. On the contrary, SAE1 silence induces an obvious suppression of the SUMOylation and Ser473 phosphorylation of Akt, which inhibits glioma cell proliferation and the tumor xenograft growth through inducing cell cycle arrest at G2 phase and cell apoptosis driven by serial biochemical molecular events. CONCLUSION: SAE1 promotes glioma cancer progression via enhancing Akt SUMOylation-mediated signaling pathway, which indicates targeting SUMOylation is a promising therapeutic strategy for human glioma.

Multifunctional Pharmaceutical Effects of the Antibiotic Daptomycin.

Daptomycin (DAP), a cyclic lipopeptide produced by Streptomyces roseosporus, is a novel antibiotic to clinically treat various Gram-positive pathogenic bacteria-induced infections. Although DAP has a strong broad-spectrum bactericidal effect, recently rare bacterial antibiotic resistance against DAP gradually arises. The review is to summarize the normal indications of DAP, its off-label usage against several clinical pathogen infections, the unique antibacterial mechanisms of DAP, and the combination of antibiotic therapies for highly DAP-resistant pathogens. More noticeably, rising evidences demonstrate that DAP has new potential activity of anticancer and immunomodulatory effects. So far the multifunctional pharmaceutical effects of DAP deserve to be further explored for future clinical applications.

MS-based strategies for identification of protein SUMOylation modification.

Protein SUMOylation modification conjugated with small ubiquitin-like modifiers (SUMOs) is one kind of PTMs, which exerts comprehensive roles in cellular functions, including gene expression regulation, DNA repair, intracellular transport, stress responses, and tumorigenesis. With the development of the peptide enrichment approaches and MS technology, more than 6000 SUMOylated proteins and about 40 000 SUMO acceptor sites have been identified. In this review, we summarize several popular approaches that have been developed for the identification of SUMOylated proteins in human cells, and further compare their technical advantages and disadvantages. And we also introduce identification approaches of target proteins which are co-modified by both SUMOylation and ubiquitylation. We highlight the emerging trends in the SUMOylation field as well. Especially, the advent of the clustered regularly interspaced short palindromic repeats/ Cas9 technique will facilitate the development of MS for SUMOylation identification.

LKB1 and YAP phosphorylation play important roles in Celastrol-induced ß-catenin degradation in colorectal cancer.

Wnt/ß-catenin and Hippo pathways play essential roles in the tumorigenesis and development of colorectal cancer. We found that Celastrol, isolated from Tripterygium wilfordii plant, exerted a significant inhibitory effect on colorectal cancer cell growth in vitro and in vivo, and further unraveled the molecular mechanisms. Celastrol induced ß-catenin degradation through phosphorylation of Yes-associated protein (YAP), a major downstream effector of Hippo pathway, and also Celastrol-induced ß-catenin degradation was dependent on liver kinase B1 (LKB1). Celastrol increased the transcriptional activation of LKB1, partially through the heat shock factor 1 (HSF1). Moreover, LKB1 activated AMP-activated protein kinase α (AMPKα) and further phosphorylated YAP, which eventually promoted the degradation of ß-catenin. In addition, LKB1 deficiency promoted colorectal cancer cell growth and attenuated the inhibitory effect of Celastrol on colorectal cancer growth both in vitro and in vivo. Taken together, Celastrol inhibited colorectal cancer cell growth by promoting ß-catenin degradation via the HSF1-LKB1-AMPKα-YAP pathway. These results suggested that Celastrol may potentially serve as a future drug for colorectal cancer treatment.