Inhibition of colon cancer K-RasG13D mutation reduces cancer cell proliferation but promotes stemness and inflammation via RAS/ERK pathway.

K-Ras is a well-studied oncogene, and its mutation is frequently found in epithelial cancers like pancreas, lung, and colorectal cancers. Cancer cells harboring K-Ras mutations are difficult to treat due to the drug resistance and metastasis properties. Cancer stem cells (CSCs) are believed the major cause of chemotherapeutic resistance and responsible for tumor recurrence and metastasis. But how K-Ras mutation affects CSCs and inflammation is not clear. Here, we compared two colon cancer cell lines, HCT-116 and HT-29, with the former being K-RasG13D mutant and the latter being wildtype. We found that HCT-116 cells treated with a K-Ras mutation inhibitor S7333 formed significantly more tumor spheroids than the untreated control, while the wild type of HT-29 cells remained unchanged. However, the size of tumor spheroids was smaller than the untreated controls, indicating their proliferation was suppressed after S7333 treatment. Consistent with this, the expressions of stem genes Lgr5 and CD133 significantly increased and the expression of self-renewal gene TGF-ß1 also increased. The flow cytometry analysis indicated that the expression of stem surface marker CD133 increased in the treated HCT-116 cells. To understand the pathway through which the G13D mutation induced the effects, we studied both RAS/ERK and PI3K/Akt pathways using specific inhibitors SCH772984 and BEZ235. The results indicated that RAS/ERK rather than PI3K/Akt pathway was involved. As CSCs play the initial role in cancer development and the inflammation is a vital step during tumor initiation, we analyzed the correlation between increased stemness and inflammation. We found a close correlation of increased Lgr5 and CD133 with proinflammatory factors like IL-17, IL-22, and IL-23. Together, our findings suggest that K-RasG13D mutation promotes cancer cell growth but decreases cancer stemness and inflammation thus tumorigenesis and metastasis potential in colon cancer. Inhibition of this mutation reverses the process. Therefore, care needs be taken when employing targeted therapies to K-RasG13D mutations in clinics.

Emodin inhibits invasion and migration of hepatocellular carcinoma cells via regulating autophagy-mediated degradation of snail and ß-catenin.

BACKGROUND: Previous studies reported that emodin extracted from Rheum palmatum L. exerts antiproliferation and antimetastatic effects in a variety of human cancer types. However, the role of emodin in hepatocellular carcinoma (HCC) remain unknown. METHODS: EdU and colony formation assays were performed to evaluate the effects of emodin on proliferation. The mobility capacities of HCC treated with emodin were evaluated using wound healing assay. Transwell invasion and migration assays were performed to evaluate anti-migratory and anti-invasive effects of emodin on HCC. Annexin V-FITC/PI was performed to analyze the apoptosis. PI stain was performed to analyze cell cycle. RNA sequencing technology was used to identify the differentially expressed genes (DEGs) induced by emodin in HCC. The impact of emodin on autophagic flux in HepG2 cells was examined by mCherry-GFP-LC3 analysis. Western blot was used to assess the protein expressions of epithelial-mesenchymal transition (EMT), autophagy, PI3K/AKT/mTOR and Wnt/ß-catenin signaling pathway. RESULTS: We found that emodin inhibited the growth of HepG2 cells in a dose- and time-dependent manner. In addition, emodin inhibited cell proliferation, induced S and G2/M phases arrest, and promoted apoptosis in HepG2 cells. The migration and invasion of HepG2 cells were also suppressed by emodin. Enrichment analysis revealed that DEGs involved in cell adhesion, cancer metastasis and cell cycle arrest. Moreover, western bolt results show that emodin-induced autophagy promotes Snail and ß-catenin degradation. We also found that blocking autophagic flux after emodin treatment caused EMT reversal. Furthermore, the PI3K agonist Y-P 740 significantly reversed the phosphorylation levels of GSK3ß and mTOR. These results indicated that emodin induced autophagy and inhibited the EMT in part through suppression of the PI3K/AKT/mTOR and Wnt/ß-catenin pathways. CONCLUSION: Our study indicated that emodin inhibited cell metastasis in HCC via the crosstalk between autophagy and EMT.

ATP stabilised and sensitised calcium phosphate nanoparticles as effective adjuvants for a DNA vaccine against cancer.

Cancer vaccines based on DNA encoding oncogenes have shown great potential in preclinical studies. However, the efficacy of DNA vaccines is limited by their weak immunogenicity because of low cellular internalisation and insufficient activation of dendritic cells (DCs). Calcium phosphate (CP) nanoparticles (NPs) are biodegradable vehicles with low toxicity and high loading capacity of DNA but suffer from stability issues. Here we employed adenosine triphosphate (ATP) as a dual functional agent, i.e. stabiliser for CP and immunological adjuvant, and applied the ATP-modified CP (ACP) NPs to the DNA vaccine. ACP NP-enhanced cellular uptake and improved transfection efficiency of DNA vaccine, and further showed the ability to activate DCs that are critical for them to prime T cells in cancer immunotherapy. As a result, a higher level of antigen-specific antibody with stronger tumour growth inhibition was achieved in mice immunised with the ACP-DNA vaccine. Overall, this one-step synthesised ACP NPs are an efficient nano-delivery system and nano-adjuvant for cancer DNA vaccines.

Prognostic and therapeutic TILs of cervical cancer-Current advances and future perspectives.

Cervical cancer is a top lethal cancer for women worldwide. Although screening and vaccination programs are available in many countries, resulting in the decline of new cases, this is not true for developing countries where there are many new cases and related deaths. Cancer immunotherapy through adaptive cell therapy (ACT) has been applied in clinics, but now much attention is focused on autogenic tumor-infiltrating lymphocyte (TIL)-based therapy, which has shown more specificity and better ability to inhibit tumor growth. Data from melanoma and cervical cancers confirm that tumor-specific T cells in TILs can be expanded for more specific and effective ACT. Moreover, TILs are derived from individual patients and are ready to home back to kill tumor cells after patient infusion, aligning well with personalized and precision medicine. In addition to therapy, TIL cell types and numbers are good indicators of host immune response to the tumor, and thus they have significant values in prognosis. Because of the special relationship with human papillomavirus (HPV) infection, cervical cancer has some specialties in TIL-based prognosis and therapy. In this review, we summarize the recent advances in the prognostic significance of TILs and TIL-based therapy for cervical cancer and discuss related perspectives.

Mannose-Functionalized Biodegradable Nanoparticles Efficiently Deliver DNA Vaccine and Promote Anti-tumor Immunity.

Cancer vaccines have attracted increasing attention for their application in tumor immunotherapy. DNA vaccines are one of them that have been proven very promising with the advantages of safety, rapid design, and low cost. However, the low stability, ineffective cell internalization, and low immunostimulation hinder their wide application. Thus, developing targeted and safe systems to effectively deliver DNA vaccines becomes a vital step. In this study, we report the development of mannose- and bisphosphonate (BP)-modified calcium phosphate (CP) nanoparticles (NPs) as efficient vaccine delivery vehicles by targeting C-type lectin receptors (CLRs) on antigen-presenting cells (APCs). Using a model antigen ovalbumin (OVA)-encoded plasmid DNA (pOVA) as a model vaccine, we demonstrate that mannose-modified and BP-stabilized CP (MBCP) nanoparticles are mono-dispersed for enhanced uptake by APCs and subsequently induce OVA antigen presentation and immunostimulation. Mice immunized with MBCP-pOVA nanovaccines show a significantly stronger anti-OVA antibody response with a quicker IgG1 and IgG2a antibody production than unmodified NPs. Moreover, MBCP-pOVA immunization significantly inhibits the growth of OVA-expressing E.G7 tumor cells in C57BL/6J mice. Our data collectively suggest that the modifications to enhance the stability and targeting ability of MBCP NPs are essential for effective delivery of DNA vaccines and promote robust anti-tumor immunity.

Clinical analysis and pluripotent stem cells-based model reveal possible impacts of ACE2 and lung progenitor cells on infants vulnerable to COVID-19.

Introduction: An increasing number of children with severe coronavirus disease 2019 (COVID-19) is being reported, yet the spectrum of disease severity and expression patterns of angiotensin-converting enzyme 2 (ACE2) in children at different developmental stages are largely unknow. Methods: We analysed clinical features in a cohort of 173 children with COVID-19 (0-15 yrs.-old) between January 22, 2020 and March 15, 2020. We systematically examined the expression and distribution of ACE2 in different developmental stages of children by using a combination of children's lung biopsies, pluripotent stem cell-derived lung cells, RNA-sequencing profiles, and ex vivo SARS-CoV-2 pseudoviral infections. Results: It revealed that infants (< 1yrs.-old), with a weaker potency of immune response, are more vulnerable to develop pneumonia whereas older children (> 1 yrs.-old) are more resistant to lung injury. The expression levels of ACE2 however do not vary by age in children's lung. ACE2 is notably expressed not only in Alveolar Type II (AT II) cells, but also in SOX9 positive lung progenitor cells detected in both pluripotent stem cell derivatives and infants' lungs. The ACE2+SOX9+ cells are readily infected by SARS-CoV-2 pseudovirus and the numbers of the double positive cells are significantly decreased in older children. Conclusions: Infants (< 1 yrs.-old) with SARS-CoV-2 infection are more vulnerable to lung injuries. ACE2 expression in multiple types of lung cells including SOX9 positive progenitor cells, in cooperation with an unestablished immune system, could be risk factors contributing to vulnerability of infants with COVID-19. There is a need to continue monitoring lung development in young children who have recovered from SARS-CoV-2 infection.

Silencing PD-1 and PD-L1 with nanoparticle-delivered small interfering RNA increases cytotoxicity of tumor-infiltrating lymphocytes.

AIM: To determine if silencing PD-1 on tumor-infiltrating lymphocytes (TILs) and its ligand-1 (PD-L1) on cancer cells will enhance the cytotoxicity of TILs. MATERIALS & METHODS: Lipid-coated calcium phosphate nanoparticles were synthesized to deliver siRNAs against PD-1 and PD-L1 to TILs and breast cancer MCF-7 cells. The downregulation of PD-1/PD-L1 expressions was determined by real-time PCR and western blotting assays. The killing efficacy of TILs to MCF-7 cells was determined by cytotoxic T lymphocyte assay. RESULTS: Lipid-coated calcium phosphate nanoparticles effectively delivered siRNAs and silenced PD-1 and PD-L1sh expression. The knockdown of either gene or both greatly improved the cytotoxicity of TILs. CONCLUSION: Silencing PD-1 and PD-L1 is an effective approach to increase TIL cytotoxicity to cancer cells.

Insluin and epithelial growth factor (EGF) promote programmed death ligand 1(PD-L1) production and transport in colon cancer stem cells.

BACKGROUND: Programmed cell death ligand 1 (PD-L1) is an important immune-inhibitory protein expressed on cancer cells to mediate cancer escape through interaction with PD-1 expressed on activated T lymphocytes (T cells). Previously, we reported that colon and breast cancer stem cells (CSCs) expressed much higher levels of PD-L1 than their parental cells, suggesting they will be more resistant to immune attack. METHODS: We investigated the underlining mechanism of PD-L1 increase in colon CSCs, with a special focus on the effect of insulin and epithelial growth factor (EGF), the two fundamental components to sustain the metabolism and stemness in the culture of CSCs. RESULTS: We found that insulin increased the total and surface PD-L1 levels through PI3K/Akt/mTOR pathway as the increase could be inhibited by the dual inhibitor of the pathway, BEZ235. EGF didn't affect the total PD-L1 levels of CSCs but increased the cell surface protein levels by flow cytometry analysis, indicating EGF promotes the transport of PD-L1 to the cell surface. Blocking cell surface PD-L1 with a specific antibody resulted in a significant reduction of tumour sphere formation but didn't interfere with the sphere growth, suggesting that cell surface PD-L1 may act as an adhering molecule for CSCs. CONCLUSIONS: Apart from the essential roles in metabolism and stemness, insulin and EGF involve in up-regulation of PD-L1 expression in colon CSCs, therefore the inhibition of insulin and EGF/EGFR pathways can be considered for cancer immunotherapy or combined with PD-1/PD-L1 antibody-based cancer immunotherapy to eliminate CSCs.

Enhancing PD-1 Gene Silence in T Lymphocytes by Comparing the Delivery Performance of Two Inorganic Nanoparticle Platforms.

Suitable carriers are crucial to RNAi applications for cancer genotherapy and T-cell immunotherapy. In this research, we selected two extensively-investigated biocompatible inorganic nanoparticle carriers, i.e., layered double hydroxide (LDH) and lipid-coated calcium phosphate (LCP) and then compared their efficacy for siRNA delivery in T cells, in order to understand which carrier is more efficient in delivering functional programmed cell death protein 1 siRNA (PD-1 siRNA) to suspended T lymphocytes. Both LDH and LCP nanoparticles quickly delivered gene segment to mouse T cell lines (EL4), while the LCP nanoparticles exhibited more cellular uptake and higher PD-1 gene silence efficiency. We further demonstrated that LCP nanoparticles successfully reduced the expression of PD-1 in human ex vivo tumor infiltrating lymphocytes (TILs). Thus, LCP nanoparticles can be used as a better nano-carrier for gene therapy in lymphocytes, especially in regards to TIL-related cancer immunotherapy.

Undo the brake of tumour immune tolerance with antibodies, peptide mimetics and small molecule compounds targeting PD-1/PD-L1 checkpoint at different locations for acceleration of cytotoxic immunity to cancer cells.

Recent clinical success of immunotherapy that inhibits the negative immune regulatory pathway programmed cell death protein-1/PD-1 ligand 1 (PD-1/PD-L1) has initiated a new era in the treatment of metastatic cancer. However, greater challenges remain to treat all cancers. The molecular architecture in the immune synapse constituting positive engagements for immune activation and negative checkpoints against immune hyperactivity is regulated dynamically by interaction between proteostasis and tumour microenvironment. This article reviews recent progresses in our understandings of the cellular and molecular mechanisms of the negative checkpoint PD-1/PD-L1 behaviours in immune tolerance of tumourigenesis and metastasis. We provide an overview on PD-L1 gene expression regulation, protein turnover, intra- and extracellular trafficking, exosome-mediated inter-cellular transport, molecular interface peptide mimetics, inhibitory chemical compounds such as metformin, and antibody dynamics. We summarise PD-L1 post-translational modifications including glycosylation, palmitoylation, phosphorylation and ubiquitination, reflecting future research directions and opportunities in identifying tumour-specific signalling targets, their regulatory molecules and pathways for intervention into various types of cancers.

Increased PD-L1 expression in breast and colon cancer stem cells.

Here we report the expression of programmed cell death ligand 1/2 (PD-L1/L2) in breast and colon cancer stem cells (CSCs). The stemness of these cells was confirmed by their surface markers. Using flow cytometry analysis we demonstrated that PD-L1 expression was higher in CSCs of both cancers compared to non-stem like cancer cells. Consistent with this, detection of cellular PD-L1 proteins by western blot assay also showed increased PD-L1 protein in CSCs. In contrast, only trace amounts of PD-L2 were detected in CSCs of both cancers. Our results suggest that breast and colon cancers may be sensitive to PD1/PD-L1 immunotherapy and thus warrant further investigations of CSC targeted PD1/PD-L1 therapy.

Comprehensive profiling of EBV gene expression in nasopharyngeal carcinoma through paired-end transcriptome sequencing.

The latent expression pattern of Epstein-Barr Virus (EBV) genes in nasopharyngeal carcinoma (NPC) has been extensively investigated, and the expression of several lytic genes in NPC has been reported. However, comprehensive information through EBV transcriptome analysis in NPC is limited. We performed paired-end RNA-seq to systematically and comprehensively characterize the expression of EBV genes in NPC tissue and C666-1 NPC cell line, which consistently carries EBV. In addition to the transcripts restricted to type II latency infection, the type III latency EBNA3s genes and a substantial number of lytic genes, such as BZLF1, BRLF1, and BMRF1, were detected through RNA-seq and were further verified in C666-1 cells and NPC tissue through realtime PCR.We also performed clustering analysis to classify NPC patient groups in terms of EBV gene expression, which presented two subtypes of NPC samples. Results revealed interesting patterns of EBV gene expression in NPC patients. This clustering was correlated with many signaling pathways, such as those related to heterotrimeric G-protein signaling, inflammation mediated by chemokine and cytokine signaling, ribosomes, protein metabolism, influenza infection, and ECM-receptor interaction. Our combined findings suggested that the expression of EBV genes in NPC is restricted not only to type II latency genes but also to type III latency and lytic genes. This study provided further insights into the potential role of EBV in the development of NPC.

Synergistic anticancer effect of rAd/P53 combined with 5-fluorouracil or iodized oil in the early therapeutic response of human colon cancer in vivo.

Exogenous wild-type p53 (wt-p53) tumor suppression increases the sensitivity of tumor cells to radiotherapy and chemotherapy. An iodized oil emulsion was used as a p53 vector for intra-arterial gene delivery to treat hepatic tumors. Whether the chemotherapeutic agent or the iodized oil affects exogenous wt-p53 activity remains poorly understood. In the present study, the early therapeutic response of rAd/p53, combined with 5-fluorouracil (5-FU) or with iodized oil, was observed in a human colon cancer model. Allograft models in 82 nude mice with human colon carcinoma SW480 were divided randomly into four groups and administered with physiologic saline, rAd/p53, rAd/p53+5-FU, and rAd/p53+iodized oil by intratumoral injection. At 24, 48, 72, 120, and 168 h after treatment, p53 expression, the Ki-67 index (KI), and the degree of tumor necrosis were assessed. The p53 expression and tumor necrosis in the therapeutic groups were higher than those in the control group. p53 expression reached its peak at 120 h in the rAd/p53 group, at 72 h in the rAd/p53+5-FU group, and at 48 h in the rAd/p53+iodized oil group. The p53 expression in the rAd/P53+5-FU group and the iodized oil group was significantly higher than those in the rAd/P53 group at 24 and 48 h. The results revealed that tumor necrosis is positively correlated with p53 expression. The KI of the rAd/p53+5-FU group increased significantly at 24 h. 5-FU and iodized oil increase the anticancer effect of rAd/p53, and 5-FU combined with rAd/p53 has a synergistic anticancer effect.

The prognostic value of tumor-infiltrating neutrophils in gastric adenocarcinoma after resection.

BACKGROUND: Several pieces of evidence indicate that tumor-infiltrating neutrophils (TINs) are correlated to tumor progression. In the current study, we explore the relationship between TINs and clinicopathological features of gastric adenocarcinoma patients. Furthermore, we investigated the prognostic value of TINs. PATIENTS AND METHODS: The study was comprised of two groups, training group (115 patients) and test group (97 patients). Biomarkers (intratumoral CD15+ neutrophils) were assessed by immunohistochemistry. The relationship between clinicopathological features and patient outcome were evaluated using Cox regression and Kaplan-Meier analysis. RESULTS: Immunohistochemical detection showed that the tumor-infiltrating neutrophils (TINs) in the training group ranged from 0.00-115.70 cells/high-power microscopic field (HPF) and the median number was 21.60 cells/HPF. Based on the median number, the patients were divided into high and low TINs groups. Chi-square test analysis revealed that the density of CD15+ TINs was positively associated with lymph node metastasis (p = 0.024), distance metastasis (p = 0.004) and UICC (International Union Against Cancer) staging (p = 0.028). Kaplan-Meier analysis showed that patients with a lower density of TINs had a better prognosis than patients with a higher density of TINs (p = 0.002). Multivariate Cox's analysis showed that the density of CD15+ TINs was an independent prognostic factor for overall survival of gastric adenocarcinoma patients. Using another 97 patients as a test group and basing on the median number of TINs (21.60 cells/HPF) coming from the training group, Kaplan-Meier analysis also showed that patients with a lower density of TINs had a better prognosis than patients with a higher density of TINs (p = 0.032). The results verify that the number of CD15+ TINs can predict the survival of gastric adenocarcinoma surgical patients. CONCLUSIONS: The presence of CD15+ TINs is an independent and unfavorable factor in the prognosis of gastric adenocarcinoma patients. Targeting CD15+ TINs may be a potential intervenient therapy in the future.

The tumor-suppressor gene Nkx2.8 suppresses bladder cancer proliferation through upregulation of FOXO3a and inhibition of the MEK/ERK signaling pathway.

Invasive bladder cancer is a lethal disease for which effective prognostic markers as well as potential therapy targets are still lacking. Nkx2.8 (Nk2 homeobox 8), a novel member of the NK-2 gene family, was reported to play an important role in the development and progression of human cancer. Herein, we reported that Nkx2.8 was markedly reduced in bladder cancer tissues compared with matched adjacent normal urothelial tissues. Nkx2.8 levels were inversely correlated with advanced T classification, N classification, tumor multiplicity, high proliferation index (Ki-67) and poor survival of patients. Furthermore, we found that overexpression of Nkx2.8 in bladder cancer cells significantly inhibited cell proliferation in vitro and in vivo, whereas silencing Nkx2.8 dramatically enhanced cell proliferation. Moreover, we demonstrated that overexpression of Nkx2.8 resulted in G(1)/S phase arrest, accompanied by upregulation of p27(Kip1), downregulation of cyclin D1 and p-FOXO3a and inhibition of MEK/ERK pathway activity. Meanwhile, silencing Nkx2.8 led to acceleration of G(1)/S transition, downregulation of p27(Kip1), upregulation of cyclin D1 and p-FOXO3a and increase of MEK/ERK pathway activity. These findings suggest that Nkx2.8 plays a potential tumor suppressor role in bladder cancer progression and represents a valuable clinical prognostic marker of this disease.

PPARγ suppression inhibits adipogenesis but does not promote osteogenesis of human mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are the common progenitors of osteoblasts and adipocytes. A reciprocal relationship exists between osteogenesis and adipogenesis in the bone marrow, and the identification of signaling pathways that stimulate MSC osteogenesis at the expense of adipogenesis is of great importance from the viewpoint of developing new therapeutic treatments for bone loss. The adipogenic transcription factor peroxisome proliferator-activated receptor γ (PPARγ) has been reported to play a vital role in modulating mesenchymal lineage allocation within the bone marrow compartment, stimulating adipocyte development at the expense of osteoblast differentiation. Hence, PPARγ may be a valuable target for drugs intended to enhance bone mass. However, little direct evidence is available for the role played by PPARγ in human mesenchymal lineage allocation. In this study, using human MSCs as an in vitro model, we showed that the two isoforms of PPARγ, PPARγ1 and PPARγ2, were differentially induced during hMSC adipogenesis, whereas only PPARγ1 was detected during osteogenesis. BADGE and GW9662, two potential antagonists of PPARγ, as well as lentivirus-mediated knockdown of PPARγ, inhibited hMSC adipogenesis but did not significantly affect osteogenesis. PPARγ knockdown did not significantly influence the expression level of the osteogenic transcription factor Runx2. Together, these results suggest that PPARγ is not the master factor regulating mesenchymal lineage determination in human bone marrow.

Astrocyte elevated gene-1 (AEG-1) is a marker for aggressive salivary gland carcinoma.

BACKGROUND: Astrocyte elevated gene-1 (AEG-1) is associated with tumorigenesis and progression in diverse human cancers. The present study was aimed to investigate the clinical and prognostic significance of AEG-1 in salivary gland carcinomas (SGC). METHODS: Real-time PCR and western blot analyses were employed to examine AEG-1 expression in two normal salivary gland tissues, eight SGC tissues of various clinical stages, and five pairs of primary SGC and adjacent salivary gland tissues from the same patient. Immunohistochemistry (IHC) was performed to examine AEG-1 protein expression in paraffin-embedded tissues from 141 SGC patients. Statistical analyses was applies to evaluate the diagnostic value and associations of AEG-1 expression with clinical parameters. RESULTS: AEG-1 expression was evidently up-regulated in SGC tissues compared with that in the normal salivary gland tissues and in matched adjacent salivary gland tissues. AEG-1 protein level was positively correlated with clinical stage (P < 0.001), T classification (P = 0.008), N classification (P = 0.008) and M classifications (P = 0.006). Patients with higher AEG-1 expression had shorter overall survival time, whereas those with lower tumor AEG-1 expression had longer survival time. CONCLUSIONS: Our results suggest that AEG-1 expression is associated with SGC progression and may represent a novel and valuable predictor for prognostic evaluation of SGC patients.