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PI3Kδ is expressed predominately in leukocytes and overexpressed in B-cell-related malignances. PI3Kδ has been validated as a promising target for cancer therapy, and specific PI3Kδ inhibitors were approved for clinical practice. However, the substantial toxicity and relatively low efficacy as a monotherapy in diffuse large B-cell lymphoma (DLBCL) limit their clinical use. In this study, we described a novel PI3Kδ inhibitor SAF-248, which exhibited high selectivity for PI3Kδ (IC 50  = 30.6 nM) over other PI3K isoforms at both molecular and cellular levels, while sparing most of the other human protein kinases in the kinome profiling. SAF-248 exhibited superior antiproliferative activity against 27 human lymphoma and leukemia cell lines compared with the approved PI3Kδ inhibitor idelalisib. In particular, SAF-248 potently inhibited the proliferation of a panel of seven DLBCL cell lines (with GI 50 values < 1 μM in 5 DLBCL cell lines). We demonstrated that SAF-248 concentration-dependently blocked PI3K signaling followed by inducing G 1 phase arrest and apoptosis in DLBCL KARPAS-422, Pfeiffer and TMD8 cells. Its activity against the DLBCL cells was negatively correlated to the protein level of PI3Kα. Oral administration of SAF-248 dose-dependently inhibited the growth of xenografts derived from Pfeiffer and TMD8 cells. Activation of mTORC1, MYC and JAK/STAT signaling was observed upon prolonged treatment and co-targeting these pathways would potentiate the activity of SAF-248. Taken together, SAF-248 is a promising selective PI3Kδ inhibitor for the treatment of DLBCL and rational drug combination would further improve its efficacy., (© 2021. The Author(s), under exclusive licence to CPS and SIMM.)

Checkpoint kinase 1 inhibitors (CHK1i) have shown impressive single-agent efficacy in treatment of certain tumors, as monotherapy or potentiators of chemotherapy in clinical trials, but the sensitive tumor types and downstream effectors to dictate the therapeutic responses to CHK1i remains unclear. In this study we first analyzed GDSC (Genomics of Drug Sensitivity in Cancer) and DepMap database and disclosed that hematologic malignancies (HMs) were relatively sensitive to CHK1i or CHK1 knockdown. This notion was confirmed by examining PY34, a new and potent in-house selective CHK1i, which exhibited potent anti-HM effect in vitro and in vivo, as single agent. We demonstrated that the downregulation of c-Myc and its signaling pathway was the common transcriptomic profiling response of sensitive HM cell lines to PY34, whereas overexpressing c-Myc could partially rescue the anticancer effect of PY34. Strikingly, we revealed the significant correlations between downregulation of c-Myc and cell sensitivity to PY34 in 17 HM cell lines and 39 patient-derived cell (PDC) samples. Thus, our results demonstrate that HMs are more sensitive to CHK1i than solid tumors, and c-Myc downregulation could represent the CHK1i efficacy in HMs., (© 2021. The Author(s), under exclusive licence to CPS and SIMM.)

A number of highly multiplexed immunostaining and imaging methods have advanced spatial proteomics of cancer for improved treatment strategies. While a variety of methods have been developed, the most widely used methods are limited by harmful signal removal techniques, difficulties with reagent production and antigen sensitivity. Multiplexed immunostaining employing oligonucleotide (oligos)-barcoded antibodies is an alternative approach that is growing in popularity. However, challenges remain in consistent conjugation of oligos to antibodies with maintained antigenicity as well as non-destructive, robust and cost-effective signal removal methods. Herein, a variety of oligo conjugation and signal removal methods were evaluated in the development of a robust oligo conjugated antibody cyclic immunofluorescence (Ab-oligo cyCIF) methodology. Both non- and site-specific conjugation strategies were assessed to label antibodies, where site-specific conjugation resulted in higher retained binding affinity and antigen-specific staining. A variety of fluorescence signal removal methods were also evaluated, where incorporation of a photocleavable link (PCL) resulted in full fluorescence signal removal with minimal tissue disruption. In summary, this work resulted in an optimized Ab-oligo cyCIF platform capable of generating high dimensional images to characterize the spatial proteomics of the hallmarks of cancer., (© 2021. The Author(s).)

Bladder cancer (BCa) is the most common malignancy of the urinary system with increasing incidence, mortality, and limited treatment options. Therefore, it is imperative to validate preclinical models that faithfully represent BCa cellular, molecular, and metabolic heterogeneity to develop new therapeutics. We performed metabolomic profiling of premalignant and non-muscle invasive bladder cancer (NMIBC) that ensued in the chemical carcinogenesis N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) mouse model. We identified the enriched metabolic signatures that associate with premalignant and NMIBC. We found that enrichment of lipid metabolism is the forerunner of carcinogen-induced premalignant and NMIBC lesions. Cross-species analysis revealed the prognostic value of the enzymes associated with carcinogen-induced enriched metabolic in human disease. To date, this is the first study describing the global metabolomic profiles associated with early premalignant and NMIBC and provide evidence that these metabolomic signatures can be used for prognostication of human disease., (© 2021. The Author(s).)

The availability of L-arginine in tumours is a key determinant of an efficient anti-tumour T cell response 1-4 . Consequently, increases of typically low L-arginine concentrations within the tumour may greatly potentiate the anti-tumour responses of immune checkpoint inhibitors, such as programmed death-ligand 1 (PD-L1)-blocking antibodies 5 . However, currently no means are available to locally increase intratumoural L-arginine levels. Here we used a synthetic biology approach to develop an engineered probiotic Escherichia coli Nissle 1917 strain that colonizes tumours and continuously converts ammonia, a metabolic waste product that accumulates in tumours 6 , to L-arginine. Colonization of tumours with these bacteria increased intratumoural L-arginine concentrations, increased the number of tumour-infiltrating T cells and had marked synergistic effects with PD-L1 blocking antibodies in the clearance of tumours. The anti-tumour effect of these bacteria was mediated by L-arginine and was dependent on T cells. These results show that engineered microbial therapies enable metabolic modulation of the tumour microenvironment leading to enhanced efficacy of immunotherapies., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Sarcomas are complex mesenchymal neoplasms with a poor prognosis. Their clinical management is highly challenging due to their heterogeneity and insensitivity to current treatments. Although there have been advances in understanding specific genomic alterations and genetic mutations driving sarcomagenesis, the underlying molecular mechanisms, which are likely to be unique for each sarcoma subtype, are not fully understood. This is in part due to a lack of consensus on the cells of origin, but there is now mounting evidence that they originate from mesenchymal stromal/stem cells (MSCs). To identify novel treatment strategies for sarcomas, research in recent years has adopted a mechanism-based search for molecular markers for targeted therapy which has included recapitulating sarcomagenesis using in vitro and in vivo MSC models. This review provides a comprehensive up to date overview of the molecular mechanisms that underpin sarcomagenesis, the contribution of MSCs to modelling sarcomagenesis in vivo, as well as novel topics such as the role of epithelial-to-mesenchymal-transition (EMT)/mesenchymal-to-epithelial-transition (MET) plasticity, exosomes, and microRNAs in sarcomagenesis. It also reviews current therapeutic options including ongoing pre-clinical and clinical studies for targeted sarcoma therapy and discusses new therapeutic avenues such as targeting recently identified molecular pathways and key transcription factors.

Partial hepatectomy (PHx) is the gold standard for the treatment of colorectal cancer liver metastases. However, after removing a substantial amount of hepatic tissue, growth factors are released to induce liver regeneration, which may promote the proliferation of liver micrometastases or circulating tumour cells still present in the patient. The aim of this study is to assess the effect of PHx on the growth of liver metastases induced by intrasplenic cell inoculation as well as on in vitro proliferation of the same cancer cell line. Liver tumours were induced in 18 WAG/RijHsd male rats, by seeding 250,000 syngeneic colorectal cancer cells (CC531) into the spleen. The left lateral lobe of the liver was mobilized and in half of the animals it was removed to achieve a 40% hepatectomy. Twenty-eight days after tumour induction, the animals were sacrificed and the liver was removed and sliced to assess the relative tumour surface area (RTSA%). CC531 cells were cultured in presence of foetal calf serum, non-hepatectomised (NRS) or hepatectomized rat serum (HRS), and their proliferation rate at 24, 48, and 72 h was measured. RTSA% was significantly higher in animals which had undergone PHx than in the controls (non-hepatectomised) (46.98 ± 8.76% vs. 18.73 ± 5.65%; p < 0.05). Analysing each lobe separately, this difference in favour of hepatectomized animals was relevant and statistically significant in the paramedian and caudate lobes. But in the right lobe the difference was scarce and not significant. In vitro, 2.5% HRS achieved stronger proliferative rates than the control cultures (10% FCS) or their equivalent of NRS. In this experimental model, a parallelism has been shown between the effect of PHx on the growth of colorectal cancer cells in the liver and the effect of the serum on those cells in vitro.

Hox genes are early determinants of cell identity along the anterior-posterior body axis across bilaterians. Several late non-homeotic functions of Hox genes have emerged in a variety of processes involved in organogenesis in several organisms, including mammals. Several studies have reported the misexpression of Hox genes in a variety of malignancies including acute myeloid leukemia. The Hox genes Dfd, Ubx, abd-A and Abd-B were overexpressed via the UAS-Gal4 system using Cg-Gal4, Lsp2-Gal4, He-Gal4 and HmlD3-Gal4 as specific drivers. Genetic interaction was tested by bringing overexpression lines in heterozygous mutant backgrounds of Polycomb and trithorax group factors. Larvae were visually scored for melanized bodies. Circulating hemocytes were quantified and tested for differentiation. Pupal lethality was assessed. Expression of Dfd, Ubx and abd-A, but not Abd-B in the hematopoietic compartment of Drosophila led to the appearance of circulating melanized bodies, an increase in cell number, cell-autonomous proliferation, and differentiation of hemocytes. Pupal lethality and melanized pseudo-tumors were suppressed in Psc 1 and esc 2 backgrounds while polycomb group member mutations Pc 1 and Su(z)12 3 and trithorax group member mutation TrlR 85 enhanced the phenotype. Dfd, Ubx and abd-A are leukemogenic. Mutations in Polycomb and trithorax group members modulate the leukemogenic phenotype. Our RNAseq of Cg-Gal4 > UAS-abd-A hemocytes may contain genes important to Hox gene induced leukemias.

The objective of this study is to investigate the signal changes and optimal scanning concentration of MRI in tumor tissues of tumor-bearing nude mice by SPIO-shRNA molecular probes. 30 BALB/c tumor-bearing nude mice were randomly divided into 5 groups with 6 mice in each group. At the given scanning time (before and 27 h after injection), the caudal vein was respectively injected with iron content of 6 mg·kg -1 , 12 mg·kg -1 , 18 mg·kg -1 , 24 mg·kg -1 , and 30 mg·kg -1 , and MR examination was simultaneously performed to measure signal intensity changes of tumor tissue and contralateral muscle tissue in each concentration group. After each examination above, the nude mice were sacrificed immediately, and the tumor and muscle tissues were removed for HE and Prussian blue staining,and observed under light microscope. Nude mice in 6 mg, 12 mg and 18 mg groups all survived after probe injection, but some nude mice died in 24 mg and 30 mg groups after probe injection or during scanning. The signal changes of T2WI and T2*WI sequences were the most obvious in MR scanning sequences. Compared with other groups, the signal intensity of the tumor tissue in 18 mg, 24 mg and 30 mg groups were most obvious (P < 0.05), while the 18 mg, 24 mg and 30 mg groups reached no statistical difference (P > 0.05 ); HE staining indicated that structural disorder of tumor tissue as well as increase of nuclear atypia. Prussian Blue staining showed that blue-stained iron particles were present in each experimental group,and the most densely distributed were in 18 mg,24 mg and 30 mg groups. Tumor tissue could be well labeled with SPIO-shRNA molecular probes, and the optimal MR scanning concentration (iron content) is 18 mg·kg -1 .

Pharmacological ascorbate (P-AscH - , high-dose, intravenous vitamin C) is cytotoxic to tumor cells in doses achievable in humans. Phase I studies in pancreatic cancer (PDAC) utilizing P-AscH - have demonstrated increases in progression free survival, suggesting a reduction in metastatic disease burden. The purpose of this study was to determine the effects of P-AscH - on metastatic PDAC. Several in vitro and in vivo mechanisms involved in PDAC metastases were investigated following treatment with P-AscH - . Serum from PDAC patients in clinical trials with P-AscH - were tested for the presence and quantity of circulating tumor cell-derived nucleases. P-AscH - inhibited invasion, basement membrane degradation, decreased matrix metalloproteinase expression, as well as clonogenic survival and viability during exposure to fluid shear stress. In vivo, P-AscH - significantly decreased formation of ascites, tumor burden over time, circulating tumor cells, and hepatic metastases. Both in vitro and in vivo findings were reversed with the addition of catalase suggesting that the effect of P-AscH - on metastatic disease is mediated by hydrogen peroxide. Finally, P-AscH - decreased CTC-derived nucleases in subjects with stage IV PDAC in a phase I clinical trial. We conclude that P-AscH - attenuates the metastatic potential of PDAC and may prove to be effective for treating advanced disease.

Ovarian cancer remains a significant challenge in women worldwide. Tumors of the high-grade serous carcinoma (HGSC) type represent the most common form of the disease. Development of new therapies for HGSC has been hampered by a paucity of preclinical models in which new drugs could be tested for target engagement and anti-tumor efficacy. Here, we systematically assessed in vivo growth of ovarian cancer cells, including six validated HGSC cell lines, in highly immunocompromised NSG mice by varying the injection site. We found that, with the exception of OVCAR3, HGSC cell lines COV318, COV362, KURAMOCHI, OVCAR4, and OVSAHO, generally demonstrate poor growth as either subcutaneous or intraperitoneal xenografts. Intrabursal injections performed with KURAMOCHI and COV362 cells did not improve tumor growth in vivo. Additional analysis revealed that OVSAHO and COV362 express moderate levels of estrogen receptor (ERα), which translated into improved growth of xenografts in the presence of 17β-Estradiol. Surprisingly, we also found that the growth of the widely used non-HGSC ovarian cell line SKOV3 could be significantly improved by estrogen supplementation. By describing successful establishment of estrogen-sensitive HGSC xenograft models, OVSAHO and COV362, this work will enable testing of novel therapies for this aggressive form of ovarian cancer.

Chalcomoracin (CMR) is a kind of Diels-Alder adduct extracted from the mulberry leaves. Recent studies showed that CMR has a broad spectrum of anticancer activities and induces paraptosis in breast cancer and prostate cancer cells. In this study, we investigated the effects of CMR against human non-small cell lung cancer cells and the underlying mechanisms. We found that CMR dose-dependently inhibited the proliferation of human lung cancer H460, A549 and PC-9 cells. Furthermore, exposure to low and median doses of CMR induced paraptosis but not apoptosis, which was presented as the formation of extensive cytoplasmic vacuolation with increased expression of endoplasmic reticulum stress markers, Bip and Chop, as well as activation of MAPK pathway in the lung cancer cells. Knockdown of Bip with siRNA not only reduced the cell-killing effect of CMR, but also decreased the percentage of cytoplasmic vacuoles in H460 cells. Moreover, CMR also increased the sensitivity of lung cancer cells to radiotherapy through enhanced endoplasmic reticulum stress. In lung cancer H460 cell xenograft nude mice, combined treatment of CMR and radiation caused greatly enhanced tumor growth inhibition with upregulation of endoplasmic reticulum stress proteins and activation of pErk in xenograft tumor tissue. These data demonstrate that the anticancer activity and radiosensitization effect of CMR result from inducing paraptosis, suggesting that CMR could be considered as a potential anticancer agent and radiation sensitizer in the future cancer therapeutics.

Norditerpenoids and dinorditerpenoids represent diterpenoids widely distributed in the genus Podocarpus with notable chemical structures and biological activities. We previously reported that nagilactone E (NLE), a dinorditerpenoid isolated from Podocarpus nagi, possessed anticancer effects against lung cancer cells in vitro. In this study we investigated the in vivo effect of NLE against lung cancer as well as the underlying mechanisms. We administered NLE (10 mg·kg -1 ·d -1 , ip) to CB-17/SCID mice bearing human lung cancer cell line A549 xenograft for 3 weeks. We found that NLE administration significantly suppressed the tumor growth without obvious adverse effects. Thereafter, RNA sequencing (RNA-seq) analysis was performed to study the mechanisms of NLE. The effects of NLE on A549 cells have been illustrated by GO and pathway enrichment analyses. CMap dataset analysis supported NLE to be a potential protein synthesis inhibitor. The inhibitory effect of NLE on synthesis of total de novo protein was confirmed in Click-iT assay. Using the pcDNA3-RLUC-POLIRES-FLUC luciferase assay we further demonstrated that NLE inhibited both cap-dependent and cap-independent translation. Finally, molecular docking revealed the low-energy binding conformations of NLE and its potential target RIOK2. In conclusion, NLE is a protein synthesis inhibitor with anticancer activity.

Secreted animal lectins of the galectin family are key players in cancer growth and metastasis. Here we show that galectin-8 (gal-8) induces the expression and secretion of cytokines and chemokines such as SDF-1 and MCP-1 in a number of cell types. This involves gal-8 binding to a uPAR/LRP1/integrin complex that activates JNK and the NFkB pathway. Cytokine and chemokine secretion, induced by gal-8, promotes migration of cancer cells toward cells treated with this lectin. Indeed, immune-competent gal-8 knockout (KO) mice express systemic lower levels of cytokines and chemokines while the opposite is true for gal-8 transgenic animals. Accordingly, gal-8 KO mice experience reduced tumor size and smaller and fewer metastatic lesions when injected with cancer cells. These results suggest the existence of a 'vicious cycle' whereby gal-8 secreted by the tumor microenvironment, promotes secretion of chemoattractants at the metastatic niche that promote further recruitment of tumor cells to that site. This study further implicate gal-8 in control of cancer progression and metastasis through its effects on the production of immunoregulatory cytokines.

Cancer stem cells (CSCs) play a central role in cancer initiation, progression, therapeutic resistance, and recurrence in patients. Here we present Capicua (CIC), a developmental transcriptional repressor, as a suppressor of CSC properties in breast cancer cells. CIC deficiency critically enhances CSC self-renewal and multiple CSC subpopulations of breast cancer cells without altering their growth rate or invasiveness. Loss of CIC relieves repression of ETV4 and ETV5 expression, consequently promoting self-renewal capability, EpCAM + /CD44 + /CD24 low/- expression, and ALDH activity. In xenograft models, CIC deficiency significantly increases CSC frequency and drives tumor initiation through derepression of ETV4. Consistent with the experimental data, the CD44 high /CD24 low CSC-like feature is inversely correlated with CIC levels in breast cancer patients. We also identify SOX2 as a downstream target gene of CIC that partly promotes CSC properties. Taken together, our study demonstrates that CIC suppresses breast cancer formation via restricting cancer stemness and proposes CIC as a potential regulator of stem cell maintenance.

Xanthatin is a natural sesquiterpene lactone purified from Xanthium strumarium L., which has shown prominent antitumor activity against a variety of cancer cells. In the current study, we investigated the effect of xanthatin on the growth of glioma cells in vitro and in vivo, and elucidated the underlying mechanisms. In both rat glioma C6 and human glioma U251 cell lines, xanthatin (1-15 μM) dose-dependently inhibited cell viability without apparent effect on the cell cycle. Furthermore, xanthatin treatment dose-dependently induced glioma cell apoptosis. In nude mice bearing C6 glioma tumor xenografts, administration of xanthatin (10, 20, 40 mg·kg -1 ·d -1 , ip, for 2 weeks) dose-dependently inhibited the tumor growth, but did not affect the body weight. More importantly, xanthatin treatment markedly increased the expression levels of the endoplasmic reticulum (ER) stress-related markers in both the glioma cell lines as well as in C6 xenografts, including glucose-regulated protein 78, C/EBP-homologous protein (CHOP), activating factor 4, activating transcription factor 6, spliced X-box binding protein-1, phosphorylated protein kinase R-like endoplasmic reticulum kinase, and phosphorylated eukaryotic initiation factor 2a. Pretreatment of C6 glioma cells with the ER stress inhibitor 4-phenylbutyric acid (4-PBA, 7 mM) or knockdown of CHOP using small interfering RNA significantly attenuated xanthatin-induced cell apoptosis and increase of proapoptotic caspase-3. These results demonstrate that xanthatin induces glioma cell apoptosis and inhibits tumor growth via activating the ER stress-related unfolded protein response pathway involving CHOP induction. Xanthatin may serve as a promising agent in the treatment of human glioma.

A critical limitation of Salmonella typhimurium (S. typhimurium) as an anti-cancer agent is the loss of their invasive or replicative activities, which results in no or less delivery of anti-cancer agents inside cancer cells in cancer therapy. Here we developed an oxytolerant attenuated Salmonella strain (KST0650) from the parental KST0649 (ΔptsIΔcrr) strain using radiation mutation technology (RMT). The oxytolerant KST0650 strain possessed 20-times higher replication activity in CT26 cancer cells and was less virulent than KST0649. Furthermore, KST0650 migrated effectively into tumor tissues in mice. KST0650 was further equipped with a plasmid harboring a spliced form of the intracellular pro-apoptotic protein sATF6, and the expression of sATF6 was controlled by the radiation-inducible recN promoter. The new strain was named as KST0652, in which sATF6 protein expression was induced in response to radiation in a dose-dependent manner. This strain was effectively delivered inside cancer cells and tumor tissues via the Salmonella type III secretion system (T3SS). In addition, combination treatment with KST0652 and radiation showed a synergistic anti-tumor effect in murine tumor model with complete inhibition of tumor growth and protection against death. In conclusion, we showed that RMT can be used to effectively develop an anti-tumor Salmonella strain for delivering anti-cancer agents inside tumors.

Disabled tumor suppressor genes and hyperactive oncogenes greatly contribute to cell fates during cancer development because of their genetic alterations such as somatic mutations. However, little is known about how tumor suppressor genes react to diverse oncogenes during tumor progression. Our previous study showed that RUNX3 inhibits invasiveness by preventing vascular endothelial growth factor secretion and suppressed endothelial cell growth and tube formation in colorectal cancer (CRC). Hedgehog signaling is crucial for the physiological maintenance and self-renewal of stem cells, and its deregulation is responsible for their tumor development. The mechanisms that inhibit this pathway during proliferation remain poorly understood. Here, we found that the tumor suppressor RUNX3 modulates tumorigenesis in response to cancer cells induced by inhibiting oncogene GLI1 ubiquitination. Moreover, we demonstrated that RUNX3 and GLI1 expression were inversely correlated in CRC cells and tissues. We observed a direct interaction between RUNX3 and GLI1, promoting ubiquitination of GLI1 at the intracellular level. Increased ubiquitination of GLI1 was induced by the E3 ligase β-TrCP. This novel RUNX3-dependent regulatory loop may limit the extent and duration of Hedgehog signaling during extension of the tumor initiation capacity. On the basis of our results, identification of agents that induce RUNX3 may be useful for developing new and effective therapies for CRC.

Neuroblastoma is the most common extra-cranial pediatric solid tumor, responsible for 13-15% of pediatric cancer death. Its intrinsic heterogeneity makes it difficult to target for successful therapy. The adaptor protein p140Cap/SRCIN1 negatively regulates tumor cell features and limits breast cancer progression. This study wish to assess if p140Cap is a key biological determinant of neuroblastoma outcome. RNAseq profiles of a large cohort of neuroblastoma patients show that SRCIN1 mRNA levels are an independent risk factor inversely correlated to disease aggressiveness. In high-risk patients, CGH+SNP microarray analysis of primary neuroblastoma identifies SRCIN1 as frequently altered by hemizygous deletion, copy-neutral loss of heterozygosity, or disruption. Functional experiments show that p140Cap negatively regulates Src and STAT3 signaling, affects anchorage-independent growth and migration, in vivo tumor growth and spontaneous lung metastasis formation. p140Cap also increases sensitivity of neuroblastoma cells to doxorubicin and etoposide treatment, as well as to a combined treatment with chemotherapy drugs and Src inhibitors. Our functional findings point to a causal role of p140Cap in curbing the aggressiveness of neuroblastoma, due to its ability to impinge on specific molecular pathways, and to sensitize cells to therapeutic treatment. This study provides the first evidence that the SRCIN1/p140Cap adaptor protein is a key player in neuroblastoma as a new independent prognostic marker for patient outcome and treatment. Altogether, these data highlight the potential clinical impact of SRCIN1/p140Cap expression in neuroblastoma tumors, in terms of reducing cytotoxic effects of chemotherapy, one of the main issues for pediatric tumor treatment.

Metastasis causes the main lethality in esophageal cancer patient. Garcinol, a natural compound extracted from Gambogic genera, is a histone acetyltransferase (HAT) inhibitor that has shown anticancer activities such as cell cycle arrest and apoptosis induction. In this study, we investigated the effects of garcinol on the metastasis of esophageal cancer in vitro and in vivo. We found that garcinol (5-15 μM) dose-dependently inhibited the migration and invasion of human esophageal cancer cell lines KYSE150 and KYSE450 in wound healing, transwell migration, and Matrigel invasion assays. Furthermore, garcinol treatment dose-dependently decreased the protein levels of p300/CBP (transcriptional cofactors and HATs) and p-Smad2/3 expression in the nucleus, thus impeding tumor cell proliferation and metastasis. Knockdown of p300 could inhibit cell metastasis, but CBP knockdown did not affect the cell mobility. It has been reported that TGF-β1 stimulated the phosphorylation of Smad2/3, which directly interact with p300/CBP in the nucleus, and upregulating HAT activity of p300. We showed that garcinol treatment dose-dependently suppressed TGF-β1-activated Smad and non-Smad pathway, inhibiting esophageal cancer cell metastasis. In a tail vein injection pulmonary metastasis mouse model, intraperitoneal administration of garcinol (20 mg/kg) or 5-FU (20 mg/kg) significantly decreased the number of lung tumor nodules and the expression levels of Ki-67, p300, and p-Smad2/3 in lung tissues. In conclusion, our study demonstrates that garcinol inhibits esophageal cancer metastasis in vitro and in vivo, which might be related to the suppression of p300 and TGF-β1 signaling pathways, suggesting the therapeutic potential of Garcinol for metastatic tumors.

The protective property of propolis across a wide spectrum of diseases has long been realized, yet the anti-tumor efficacy of this bioactive substance from Philippine stingless bees has remained poorly understood. Here, we showed the tumor-suppressing potential of crude ethanolic extract of Philippine stingless bee propolis (EEP) in in vitro models of gastric cancer highlighting the first indication of remarkable subtype specificity towards differentiated-type human gastric cancer cell lines but not the diffuse-type. Mechanistically, this involved the profound modulation of several cell cycle related gene transcripts, which correlated with the prominent cell cycle arrest at the G0/G1 phase. To reinforce our data, a unique differentiated-type gastric cancer model, A4gnt KO mice, together with age-matched 60 week-old C57BL/6 J mice were randomly assigned to treatment groups receiving distilled water or EEP for 30 consecutive days. EEP treatment induced significant regression of gross and histological lesions of gastric pyloric tumors that consistently corresponded with specific transcriptional regulation of cell cycle components. Also, the considerable p21 protein expression coupled with a marked reduction in rapidly dividing BrdU-labeled S-phase cells unequivocally supported our observation. Altogether, these findings support the role of Philippine stingless bee propolis as a promising adjunct treatment option in differentiated-type gastric cancer.

Pelvic recurrence of colorectal cancer is a crucial problem because radical surgery can lead to excessive invasion. Novel therapeutic strategies are required instead of surgery. However, there are few suitable models because of the difficulty in transplanting and observing tumors in the pelvis. We have established an appropriate injection site suitable for the establishment of colorectal cancer pelvic recurrence that allows for the observation of tumor growth. DLD-1 cells stably expressing luciferase (DLD-1 clone#1-Luc) were inoculated into various points of female BALB/c nude mice and the engrafted cells were analyzed with an imaging system employing bioluminescent signals and computed tomography. Weekly analysis with the imaging system showed that a triangular area defined by the vagina, the anus, and the ischial spine was suitable for the engraftment of pelvic tumors. The imaging system was able to detect the engrafted tumor 7 days after the inoculation of cells. Weight loss was observed in our model, and overall survival was 21-42 days. Tumor involvement of adjacent organs was detected histopathologically, as is the case in the clinical situation. These findings suggest that this model is valid for evaluations of the therapeutic effects of novel treatments under development. It is hoped that this model will be used in preclinical research.

Pancreatic ductal adenocarcinoma (PDAC) is supported by a complex microenvironment whose physical contribution to chemoresistance could be overcome by ultrasound (US) therapy. This study aims to investigate the ability of US-induced inertial cavitation in association with chemotherapy to alter tumor cell viability via microenvironment disruption. For this purpose, we used a 3D-coculture PDAC model partially mimicking the tumor and its microenvironment. Coculture spheroids combining DT66066 cells isolated from KPC-transgenic mice and murine embryonic fibroblasts (iMEF) were obtained by using a magnetic nanoshuttle method. Spheroids were exposed to US with incremental inertial cavitation indexes. Conditions studied included control, gemcitabine, US-cavitation and US-cavitation + gemcitabine. Spheroid viability was assessed by the reduction of resazurin and flow cytometry. The 3D-coculture spheroid model incorporated activated fibroblasts and produced type 1-collagen, thus providing a partial miniature representation of tumors with their microenvironment. Main findings were: (a) Gemcitabine (5 μM) was significantly less cytotoxic in the presence of KPC/iMEFs spheroids compared with KPC (fibroblast-free) spheroids; (b) US-induced inertial cavitation combined with Gemcitabine significantly decreased spheroid viability compared to Gemcitabine alone; (c) both cavitation and chemotherapy affected KPC cell viability but not that of fibroblasts, confirming the protective role of the latter vis-à-vis tumor cells. Gemcitabine toxicity is enhanced when cocultured spheroids of KPC and iMEF are exposed to US-cavitation. Although the model used is only a partial representation of PDAC, this experience supports the hypothesis that US-inertial cavitation can enhance drug penetration and cytotoxicity by disrupting PDAC microenvironment.

Effective drug delivery is restricted by pathophysiological barriers in solid tumors. In human pancreatic adenocarcinoma, poorly-permeable blood vessels limit the intratumoral permeation and penetration of chemo or nanotherapeutic drugs. New and clinically viable strategies are urgently sought to breach the neoplastic barriers that prevent effective drug delivery. Here, we present an original idea to boost drug delivery by selectively knocking down the tumor vascular barrier in a human pancreatic cancer model. Clinical radiation activates the tumor endothelial-targeted gold nanoparticles to induce a physical vascular damage due to the high photoelectric interactions. Active modulation of these tumor neovessels lead to distinct changes in tumor vascular permeability. Noninvasive MRI and fluorescence studies, using a short-circulating nanocarrier with MR-sensitive gadolinium and a long-circulating nanocarrier with fluorescence-sensitive nearinfrared dye, demonstrate more than two-fold increase in nanodrug delivery, post tumor vascular modulation. Functional changes in altered tumor blood vessels and its downstream parameters, particularly, changes in K trans (permeability), K ep (flux rate), and V e (extracellular interstitial volume), reflect changes that relate to augmented drug delivery. The proposed dual-targeted therapy effectively invades the tumor vascular barrier and improve nanodrug delivery in a human pancreatic tumor model and it may also be applied to other nonresectable, intransigent tumors that barely respond to standard drug therapies.

Light-activated theranostics offer promising opportunities for disease diagnosis, image-guided surgery, and site-specific personalized therapy. However, current fluorescent dyes are limited by low brightness, high cytotoxicity, poor tissue penetration, and unwanted side effects. To overcome these limitations, we demonstrate a platform for optoelectronic tuning, which allows independent control of the optical properties from the electronic properties of fluorescent organic salts. This is achieved through cation-anion pairing of organic salts that can modulate the frontier molecular orbital without impacting the bandgap. Optoelectronic tuning enables decoupled control over the cytotoxicity and phototoxicity of fluorescent organic salts by selective generation of mitochondrial reactive oxygen species that control cell viability. We show that through counterion pairing, organic salt nanoparticles can be tuned to be either nontoxic for enhanced imaging, or phototoxic for improved photodynamic therapy.

Argonaute 2 (AGO2), the core component of microRNA (miRNA)-induced silencing complex, plays a compelling role in tumorigenesis and aggressiveness. However, the mechanisms regulating the functions of AGO2 in cancer still remain elusive. Herein, we indentify one intronic circular RNA (circRNA) generated from AGO2 gene (circAGO2) as a novel regulator of AGO2-miRNA complexes and cancer progression. CircAGO2 is up-regulated in gastric cancer, colon cancer, prostate cancer, and neuroblastoma, and is associated with poor prognosis of patients. CircAGO2 promotes the growth, invasion, and metastasis of cancer cells in vitro and in vivo. Mechanistic studies reveal that circAGO2 physically interacts with human antigen R (HuR) protein to facilitate its activation and enrichment on the 3'-untranslated region of target genes, resulting in reduction of AGO2 binding and repression of AGO2/miRNA-mediated gene silencing associated with cancer progression. Pre-clinically, administration of lentivirus-mediated short hairpin RNA targeting circAGO2 inhibits the expression of downstream target genes, and suppresses the tumorigenesis and aggressiveness of xenografts in nude mice. In addition, blocking the interaction between circAGO2 and HuR by cell-penetrating inhibitory peptide represses the tumorigenesis and aggressiveness of cancer cells. Taken together, these results indicate that oncogenic circAGO2 drives cancer progression through facilitating HuR-repressed functions of AGO2-miRNA complexes.

Many acute myeloid leukaemias (AMLs) express high levels of BCL-2 and MCL-1, especially after therapy. To test the impact of these anti-apoptotic proteins on AML development and treatment, we used haemopoietic reconstitution to generate MLL-AF9 AMLs expressing BCL-2 or Mcl-1 transgenes. AMLs with elevated BCL-2 or MCL-1 had a higher proportion of mature myeloid cells but, like conventional MLL-AF9 AMLs, were readily transplantable. Short-term cell lines established from multiple primary AMLs of each genotype were tested in vitro for susceptibility to chemotherapeutics currently used for treating AML (daunorubicin, etoposide, cytarabine); the proteasome inhibitor bortezomib; CDK7/9 inhibitors; and BH3 mimetics, which bind and inhibit pro-survival proteins. The BH3 mimetics tested, alone and in combination with the other drugs, were: ABT-737 which, like its clinical counterpart navitoclax, targets BCL-2, BCL-X L and BCL-W; BCL-2-specific ABT-199 (venetoclax); BCL-X L -specific A-1331852; and S63845, a new MCL-1-specific BH3 mimetic. As single agents, daunorubicin and bortezomib had the greatest efficacy. Elevated MCL-1 or BCL-2 reduced sensitivity to daunorubicin but, surprisingly, not to bortezomib. MCL-1 markedly enhanced resistance to ABT-737 and ABT-199 but not S63845, and BCL-2 increased resistance to S63845 but not to ABT-737 or ABT-199. Notable synergies were achieved by combining BH3 mimetics with daunorubicin: S63845 increased the sensitivity of both MCL-1 and BCL-2 overexpressing MLL-AF9 AMLs, and ABT-737 aided in killing those overexpressing BCL-2. Synergy between daunorubicin and ABT-199 was also apparent in vivo, although not curative. Impressive synergistic responses were achieved for human MLL-fusion AML cell lines treated with daunorubicin plus either ABT-737, ABT-199 or S63845, and with ABT-199 plus S63845, with or without daunorubicin. Our data suggest that AML patients may benefit from combining conventional cytotoxic drugs with BH3 mimetics targeting BCL-2 or MCL-1 or, if tolerated, both these agents.

The Near-infrared Fluorescence (NIRF) molecular imaging of cancer is known to be superior in sensitivity, deeper penetration, and low phototoxicity compared to other imaging modalities. In view of an increased need for efficient and targeted imaging agents, we synthesized a NAD(P)H quinone oxidoreductase 1 (NQO1)-activatable NIR fluorescent probe (NIR-ASM) by conjugating dicyanoisophorone (ASM) fluorophore with the NQO1 substrate quinone propionic acid (QPA). The probe remained non-fluorescent until activation by NQO1, whose expression is largely limited to malignant tissues. With a large Stokes shift (186 nm) and a prominent near-infrared emission (646 nm) in response to NQO1, NIR-ASM was capable of monitoring NQO1 activity in vitro and in vivo with high specificity and selectivity. We successfully employed the NIR-ASM to differentiate cancer cells from normal cells based on NQO1 activity using fluorescence microscopy and flow cytometry. Chemical and genetic approaches involving the use of ES936, a specific inhibitor of NQO1 and siRNA and gene transfection procedures unambiguously demonstrated NQO1 to be the sole target activating the NIR-ASM in cell cultures. NIR-ASM was successfully used to detect and image the endogenous NQO1 in three live tumor-bearing mouse models (A549 lung cancer, Lewis lung carcinoma, and MDMAMB 231 xenografts) with a high signal-to-low noise ratiometric NIR fluorescence response. When the NQO1-proficient A549 tumors and NQO1-deficient MDA-MB-231 tumors were developed in the same animal, only the A549 malignancies activated the NIR-ASM probe with a strong signal. Because of its high sensitivity, rapid activation, tumor selectivity, and nontoxic properties, the NIR-ASM appears to be a promising agent with clinical applications.

Tumor-targeted drug delivery systems (Tt-DDSs) are proposed as a promising strategy for cancer care. However, the dense collagen network in tumors stroma significantly reduces the penetration and efficacy of Tt-DDS. In order to investigate the effect of asiatic acid (AA) on antitumor effect of pegylated liposomal doxorubicin (PLD) by attenuating stroma-collagen, colon cancer xenograft mice (SW620 cell line) were treated by PLD, AA, or combined regimes, respectively; the collagen levels were estimated by Sirius red/fast green dual staining and immunohistochemistry (IHC) staining; the intratumor exposure of doxorubicin was visualized by ex vivo fluorescence imaging and quantified by HPLC/MS analysis. In addition, the impact of AA on collagen synthesis of fibroblast cell (HFL-1) and cytotoxic effect of PLD and doxorubicin to cancer cell (SW620) were studied in vitro. In the presence of AA (4 mg/kg), the intratumor collagen level was restricted in vivo (reduced by 22%, from 4.14% ± 0.30% to 3.24% ± 0.25%, P = 0.051) and in vitro. Subsequently, doxorubicin level was increased by ~30%. The antitumor activity of PLD was significantly improved (57.3% inhibition of tumor growth and 44% reduction in tumor weight) by AA combination. Additionally, no significant improvement in cytotoxic effect of PLD or doxorubicin induced by AA was observed. In conclusion, AA is a promising sensitizer for tumor treatment by enhancing intratumor drug exposure via stromal remodeling.

Application of Toll-like receptor (TLR) agonists is a promising approach to treat cancer. In particular, nucleic acid-based TLR agonists such as short ssRNA and dsRNA molecules, which activate endosomal TLRs, can be delivered to tumors by use of nanoparticle delivery systems. However, such delivery systems bear unspecific side effects and poor pharmacokinetics. To overcome these limitations we developed a system for targeted delivery of a 50 bp dsRNA TLR3 agonist (Riboxxol) to treat PSCA-positive tumor cells, which consists of neutravidin conjugated to mono-biotinylated dsRNA and to humanized mono-biotinylated anti-PSCA single chain antibody derivative scFv(h-AM1)-BAP. The assembly of the components resulted in the formation of nanoparticle-like immunoconjugates designated Rapid Inducer of Cellular Inflammation and Apoptosis (RICIA). Anti-PSCA-RICIA exclusively delivered Riboxxol to PSCA-positive tumor cells as well as subcutaneous tumors. Uptake of anti-PSCA-RICIA induced a type I-interferon response and apoptosis in HEK-Blue hTLR3/PSCA reporter cells and PSCA-positive HT1376 bladder cancer cells in vitro. No such effects were observed when using RICIA coupled to an unspecific control antibody or when using Riboxxol alone. Treatment of HT1376 xenografts in immune-deficient hosts with targeted delivery of TLR3 agonist did not induce adverse effects and only modestly inhibited tumor growth when compared to controls. These results suggest promising activation of innate immune response and apoptosis upon selective delivery of TLR3 agonists in tumor cells. Yet, further studies using syngeneic and orthotopic tumor models are needed to fully exploit the potential of RICIA immunoconjugates.

The von Hippel-Lindau (VHL) syndrome is a rare inherited cancer, caused by mutations in the VHL gene, many of which render the VHL protein (pVHL) unstable. pVHL is a tumor-suppressor protein implicated in a variety of cellular processes, most notably in response to changes in oxygen availability, due to its role as part of an E3-ligase complex which targets the hypoxia-inducible factor (HIF) for degradation. Previously we reported, using in silico and in vitro analyses, that common oncogenic VHL mutations render pVHL less stable than the wild-type protein, distort its core domain and as a result reduce the ability of the protein to bind its target HIF-1α. Among various chemical chaperones tested, arginine was the most effective in refolding mutant of pVHL. Here we examined the consequences of administering L- or D-arginine to a Drosophila VHL model and to human renal carcinoma cells, both expressing misfolded versions of human pVHL. Arginine treatment increased pVHL solubility in both models and increased the half-life of the mutant pVHL proteins in the cell culture. In both models, L- as well as D-arginine enhanced the ability of wild-type pVHL and certain misfolded mutant versions of pVHL to bind ODD, the HIF-derived target peptide, reflecting restoration of pVHL function. Moreover, continuous feeding of Drosophila expressing misfolded versions of pVHL either L- or D-arginine rich diet rescued their lethal phenotype. Collectively, these in vivo results suggest that arginine supplementation should be examined as a potential novel treatment for VHL cancer syndrome.

Melanoma is the deadliest form of skin cancer with rising incidence, creating a significant health problem. We discovered increased expression of bone morphogenetic protein 6 (BMP6) in melanoma cells and tissues, and observed that BMP6 deficiency caused significantly delayed tumor onset and decelerated tumor progression in a melanoma mouse model. Moreover, we determined that BMP6 inhibits dermal mast cell recruitment and found that mast cell-derived mediators significantly reduced melanoma growth in vitro. In line with this, mast cell deficiency accelerated tumor onset and progression in a melanoma mouse model. Analysis of human melanoma tissues revealed a strong negative correlation between melanoma proliferation and mast cell infiltration. This study elucidates a novel role of BMP6-induced modulation of the tumor microenvironment.

Antrodia cinnamomea, a well-known traditional medicine used in Taiwan, is a potent anticancer drug for colorectal cancer, but the upstream molecular mechanism of its anticancer effects remains unclear. In this study, A. cinnamomea extracts showed cytotoxicity in HCT116, HT29, SW480, Caco-2 and, Colo205 colorectal cancer cells. Whole-genome expression profiling of A. cinnamomea extracts in HCT116 cells was performed. A. cinnamomea extracts upregulated the expression of the endoplasmic reticulum stress marker CHOP and its downstream gene TRB3. Moreover, dephosphorylation of Akt and mTOR as well as autophagic cell death were observed. Gene expression and autophagic cell death were reversed by the knockdown of CHOP and TRB3. Autophagy inhibition but not apoptosis inhibition reversed A. cinnamomea-induced cell death. Finally, we demonstrated that A. cinnamomea extracts significantly suppressed HCT116 tumour growth in nude mice. Our findings suggest that autophagic cell death via the CHOP/TRB3/Akt/mTOR pathway may represent a new mechanism of anti-colorectal cancer action by A. cinnamomea. A. cinnamomea is a new CHOP activator and potential drug that can be used in colorectal cancer treatment.

Chemotherapy induces tumor cell death by directly damaging DNA or hindering cell mitosis. Some of the drawbacks of most chemotherapy are lack of target selectivity to tumor cells, and adverse drug reaction, which limit the treatment intensity and frequency. Herein, we synthesized the prodrug of triptolide (TP) coupled to vitamin E (VE) using dithiodiglycolic acid and co-dissolved with PEG2000- linoleic acid (MPEG200-LD) in ethanol. The PEGylated TP prodrug self-assembly nanoparticles (PTPPSN) were prepared via nanoprecipitation method. Besides, characterization, stability and in vitro release of the PEGylated nanometer prodrug were investigated. Furthermore, in vitro and in vivo antitumor efficacy of PTPPSN explored showed that the cytotoxicity of triptolide was significantly reduced in vitro preparation. However, in vitro and in vivo antitumor effect of PTPPSN was significantly improved compared to the original triptolide. In summary, the PEGylated nanoparticle successfully encapsulated triptolide yielded suitable cell microenvironment, and nanotechnology-related achievements. This study, therefore, provides a new method for antitumor research as well as an innovative technology for clinical treatment of malignant tumor.

The excitement surrounding the potential of gene therapy has been tempered due to the challenges that have thus far limited its successful implementation in the clinic such as issues regarding stability, transfection efficiency, and toxicity. In this study, low molecular weight linear polyethyleneimine (2.5 kDa) was modified by conjugation to a lipid, lithocholic acid, and complexed with a natural polysaccharide, dermatan sulfate (DS), to mask extra cationic charges of the modified polymer. In vitro examination revealed that these modifications improved complex stability with plasmid DNA (pDNA) and transfection efficiency. This novel ternary polyplex (pDNA/3E/DS) was used to investigate if tumor-targeted radiotherapy led to enhanced accumulation and retention of gene therapy vectors in vivo in tumor-bearing mice. Imaging of biodistribution revealed that tumor irradiation led to increased accumulation and retention as well as decreased off-target tissue buildup of pDNA in not only pDNA/3E/DS, but also in associated PEI-based polyplexes and commercial DNA delivery vehicles. The DS-containing complexes developed in this study displayed the greatest increase in tumor-specific pDNA delivery. These findings demonstrate a step forward in nucleic acid vehicle design as well as a promising approach to overall cancer gene therapy through utilization of radiotherapy as a tool for enhanced delivery.

Salmonella typhimurium (hereafter S. typhimurium), as Gram-negative facultative anaerobic bacteria, are good candidates for cancer therapy and delivering therapeutic antitumor agents. However, it is necessary to reduce the virulence of such bacteria and enhance their tumor-targeting ability, and their immunostimulatory ability to induce tumor cell apoptosis. In this study, we constructed a S. typhimurium mutant named S634 harboring aroA mutation and additional mutations involved in modifications of lipid A. Upon intraperitoneal infection in mice, the aroA-deficient strain S634 showed greatly attenuated virulence and preferential accumulation within tumor tissue. We next investigated the ability of S636, the asd mutant derivative of S634, to deliver the anti-angiogenic agent "endostatin" (S636/pES) and to inhibit tumor growth in mouse CT26 colon carcinoma and B16F10 melanoma models. S636/pES-treated tumor-bearing mice showed suppressed tumor growth and prolonged survival, compared to mice treated with either the bacteria carrying empty plasmids or PBS intraperitoneally. Immunohistochemical studies demonstrated that, when tumor-bearing mice were infected with S636/pES, Salmonella colonization and endostatin expression were accompanied by the increase of apoptosis level and suppression of tumor angiogenesis within tumor tissues. Our findings showed that endostatin gene therapy delivered by attenuated S. typhimurium displays therapeutic antitumor effects in murine tumor models.

Current treatments for inflammatory bowel disease (IBD) target the overactive immune response of the intestinal mucosa. However, epidermal growth factor (EGF), an activating ligand of the EGF receptor (EGFR), has been shown to induce disease remission through direct targeting of intestinal mucosal healing. Despite promising preclinical and clinical results, this EGFR-activating therapy has not progressed, in part due to the potential for carcinogenesis associated with long-term use and the increased risk of colitis-associated cancer (CAC) in IBD. Here we tested whether pharmacological modulation of EGFR altered outcomes of CAC in the murine azoxymethane/dextran sulfate sodium model. We found that administering EGF during the period of maximum colitis severity ("early"), coincident with the initiation and early promotion of tumors, improved outcomes of colitis and reduced tumor size. In contrast, daily EGF administration beginning ~2 months after tumor initiation ("late") increased tumor size. Administration of the EGFR kinase inhibitor gefitinib increased the tumor size when the drug was given early and decreased the tumor size when the drug was administered late. EGF administration not only reduced colonic cytokine and chemokine expression during injury, but also baseline chemokine expression in homeostasis. These results suggest that EGFR activation during acute bouts of colitis may reduce the long-term burden of CAC.

There is still an unmet need for xenotransplantation models that efficiently recapitulate normal and malignant human hematopoiesis. Indeed, there are a number of strategies to generate humanized mice and specific protocols, including techniques to optimize the cytokine environment of recipient mice and drug alternatives or complementary to the standard conditioning regimens, that can be significantly modulated. Unfortunately, the high costs related to the use of sophisticated mouse models may limit the application of these models to studies that require an extensive experimental design. Here, using an affordable and convenient method, we demonstrate that the administration of fludarabine (Fludara TM ) promotes the extensive and rapid engraftment of human normal hematopoiesis in immunodeficient mice. Quantification of human CD45 + cells in bone marrow revealed approximately a 10 2 -fold increase in mice conditioned with irradiation plus fludarabine. Engrafted cells in the bone marrow included hematopoietic stem cells, as well as myeloid and lymphoid cells. Moreover, this model proved to be sufficient for robust reconstitution of malignant myeloid hematopoiesis, permitting primary acute myeloid leukemia cells to engraft as early as 8 weeks after the transplant. Overall, these results present a novel and affordable model for engraftment of human normal and malignant hematopoiesis in immunodeficient mice.

MYC-driven Group 3 (G3) medulloblastoma (MB) is the most aggressive of four molecular subgroups classified by transcriptome, genomic landscape and clinical outcomes. Mouse models that recapitulate human G3 MB all rely on retroviral vector-induced Myc expression driven by viral regulatory elements (Retro-Myc tumors). We used nuclease-deficient CRISPR/dCas9-based gene activation with combinatorial single guide RNAs (sgRNAs) to enforce transcription of endogenous Myc in Trp53-null neurospheres that were orthotopically transplanted into the brains of naïve animals. Three combined sgRNAs linked to dCas9-VP160 induced cellular Myc expression and large cell anaplastic MBs (CRISPR-Myc tumors) which recapitulated the molecular characteristics of mouse and human G3 MBs. The BET inhibitor JQ1 suppressed MYC expression in a human G3 MB cell line (HD-MB03) and CRISPR-Myc, but not in Retro-Myc MBs. This G3 MB mouse model in which Myc expression is regulated by its own promoter will facilitate pre-clinical studies with drugs that regulate Myc transcription.

To develop effective therapies for advanced high grade serous ovarian cancer (HGSOC), understanding mechanisms of recurrence and metastasis is necessary. In this study, we define the epithelial/mesenchymal status of cell lines that accurately model HGSOC, and evaluate the therapeutic potential of targeting Snai1 (Snail), a master regulator of the epithelial/mesenchymal transition (EMT) in vitro and in vivo. The ratio of Snail to E-cadherin (S/E index) at RNA and protein levels was correlated with mesenchymal morphology in four cell lines. The cell lines with high S/E index (OVCAR8 and COV318) showed more CSC-like, motile, and chemoresistant phenotypes than those with low S/E index (OVSAHO and Kuramochi). We tested the role of Snail in regulation of malignant phenotypes including stemness, cell motility, and chemotherapy resistance: shRNA-mediated knockdown of Snail reversed these malignant phenotypes. Interestingly, the expression of let-7 tumour suppressor miRNA was upregulated in Snail knockdown cells. Furthermore, knockdown of Snail decreased tumour burden in an orthotopic xenograft mouse model. We conclude that Snail is important in controlling HGSOC malignant phenotypes and suggest that the Snail/Let-7 axis may be an attractive target for HGSOC treatment.

The purpose of this study was to determine if localized delivery of IL-12 encoded by a replication-incompetent adenoviral vector engineered to express IL-12 via a RheoSwitch Therapeutic System ® (RTS ® ) gene switch (Ad-RTS-IL-12) administered intratumorally which is inducibly controlled by the oral activator veledimex is an effective approach for glioma therapy. Mice bearing 5-10-day-old intracranial GL-261 gliomas were intratumorally administered Ad-RTS-mIL-12 in which IL-12 protein expression is tightly controlled by the activator ligand, veledimex. Local tumor viral vector levels concomitant with veledimex levels, IL-12-mRNA expression, local and systemic cytokine expression, tumor and systemic flow cytometry and overall survival were studied. Ad-RTS-mIL-12+veledimex elicited a dose-related increase in tumor IL-12 mRNA and IL-12 protein and discontinuation of veledimex resulted in a return to baseline levels. These changes correlated with local immune and antitumor responses. Veledimex crossed the blood-brain barrier in both orthotopic GL-261 mice and cynomolgus monkeys. We have demonstrated that this therapy induced localized controlled production of IL-12 which correlates with an increase in tumor-infiltrating lymphocytes (TILs) leading to the desired biologic response of tumor growth inhibition and regression. At day 85 (study termination), 65% of the animals that received veledimex at 10 or 30 mg/m 2 /day were alive and tumor free. In contrast, the median survival for the other groups were: vehicle 23 days, bevacizumab 20 days, temozolomide 33 days and anti-PD-1 37 days. These findings suggest that the controlled intratumoral production of IL-12 induces local immune cell infiltration and improved survival in glioma, thereby demonstrating that this novel regulated immunotherapeutic approach may be an effective form of therapy for glioma.

Colorectal cancer (CRC) is a highly heterogeneous disease both from a molecular and clinical perspective. Several distinct molecular entities, such as microsatellite instability (MSI), have been defined that make up biologically distinct subgroups with their own clinical course. Recent data indicated that CRC can be best segregated into four groups called consensus molecular subtypes (CMS1-4), each of which has a unique biology and gene expression pattern. In order to develop improved, subtype-specific therapies and to gain insight into the molecular wiring and origin of these subtypes, reliable models are needed. This study was designed to determine the heterogeneity and identify the presence of CMSs in a large panel of CRC cell lines, primary cultures and patient-derived xenografts (PDX). We provide a repository encompassing this heterogeneity and moreover describe that a large part of the models can be robustly assigned to one of the four CMSs, independent of the stromal contribution. We subsequently validate our CMS stratification by functional analysis which for instance shows mesenchymal enrichment in CMS4 and metabolic dysregulation in CMS3. Finally, we observe a clear difference in sensitivity to chemotherapy-induced apoptosis, specifically between CMS2 and CMS4. This relates to the in vivo efficacy of chemotherapy, which delays outgrowth of CMS2, but not CMS4 xenografts. Combined our data indicate that molecular subtypes are faithfully modelled in CRC cell cultures and PDXs, representing tumour cell intrinsic and stable features. This repository provides researchers with a platform to study CRC using the existing heterogeneity.

Targeting the autophagic process is considered a promising therapeutic strategy in cancer since a great number of tumors, including melanoma, show high basal levels of protective autophagy that contributes to tumor progression and chemoresistance. Here, exploiting both in vitro and in vivo approaches, we identified N6-isopentenyladenosine (iPA), an end product of the mevalonate pathway, as a novel autophagy inhibitor with an interesting anti-melanoma activity. iPA, after being phosphorylated by adenosine kinase into 5'-iPA-monophosphate, induces autophagosome accumulation through AMPK activation, measured by increased fluorescent GFP-LC3 puncta and enhanced conversion into the lipidated autophagosome-associated LC3-II. However, at a later stage iPA blocks the autophagic flux monitored by p62 accumulation, Luciferase reporter-based assay for LC3 turnover in living cells and fluorescence of a tandem RFP-GFP-LC3 construct. Impaired autophagic flux is due to the block of autophagosome-lysosome fusion through the defective localization and function of Rab7, whose prenylation is inhibited by iPA, resulting in a net inhibition of autophagy completion that finally leads to melanoma apoptotic cell death. AMPK silencing prevents apoptosis upon iPA treatment, whereas basal autophagosome turnover is still inhibited due to unprenylated Rab7. These results strongly support the advantage of targeting autophagy for therapeutic gain in melanoma and provide the preclinical rational to further investigate the antitumor action of iPA, able to coordinately induce autophagosome accumulation and inhibit the autophagic flux, independently targeting AMPK and Rab7 prenylation. This property may be particularly useful for the selective killing of tumors, like melanoma, that frequently develop chemotherapy resistance due to protective autophagy activation.

The transcription factor p53 is a vital tumor suppressor. Upon activation by diverse stresses including oncogene activation, DNA damage, hypoxia and nutrient deprivation, p53 activates a panoply of target genes and orchestrates numerous downstream responses that suppress tumorigenesis. Although early studies of p53 suggested that its ability to induce cell cycle arrest, senescence and apoptosis programs accounted for its tumor-suppressor activity, more recent studies have challenged this notion. Moreover, p53 regulates a suite of additional processes, such as metabolism, stem cell function, invasion and metastasis. The processes p53 coordinately regulates to enact tumor suppression, and how such regulation occurs, thus remain elusive. In this review, we will summarize our current knowledge of p53-mediated tumor-suppressive mechanisms gleaned from in vivo studies in mouse models.

Hyperactivation of Wnt/β-catenin signaling is one of the major causes of human colorectal cancer (CRC). A hallmark of Wnt signaling is the nuclear accumulation of β-catenin. Although β-catenin nuclear import and export have been widely investigated, the underlying mechanism of β-catenin's nuclear retention remains largely unknown. Here, we report that Twa1/Gid8 is a key nuclear retention factor for β-catenin during Wnt signaling and colorectal carcinogenesis. In the absence of Wnt, Twa1 exists together with β-catenin in the Axin complex and undergoes ubiquitination and degradation. Upon Wnt signaling, Twa1 translocates into the nucleus, where it binds and retains β-catenin. Depletion of Twa1 attenuates Wnt-stimulated gene expression, dorsal development of zebrafish embryos and xenograft tumor growth of CRC cells. Moreover, nuclear Twa1 is significantly upregulated in human CRC tissues, correlating with the nuclear accumulation of β-catenin and poor prognosis. Thus, our results identify Twa1 as a previously undescribed regulator of the Wnt pathway for promoting colorectal tumorigenesis by facilitating β-catenin nuclear retention.

Hispidulin, a polyphenolic flavonoid extracted from the traditional Chinese medicinal plant S involucrata, exhibits anti-tumor effects in a wide array of human cancer cells, mainly through growth inhibition, apoptosis induction and cell cycle arrest. However, its precise anticancer mechanisms remain unclear. In this study, we investigated the molecular mechanisms that contribute to hispidulin-induced apoptosis of human clear-cell renal cell carcinoma (ccRCC) lines Caki-2 and ACHN. Hispidulin (10, 20 μmol/L) decreased the viability of ccRCC cells in dose- and time-dependent manners without affecting that of normal tubular epithelial cells. Moreover, hispidulin treatment dose-dependently increased the levels of cleaved caspase-8 and caspase-9, but the inhibitors of caspase-8 and caspase-9 only partly abrogated hispidulin-induced apoptosis, suggesting that hispidulin triggered apoptosis via both extrinsic and intrinsic pathways. Moreover, hispidulin treatment significantly inhibited the activity of sphingosine kinase 1 (SphK1) and consequently promoted ceramide accumulation, thus leading to apoptosis of the cancer cells, whereas pretreatment with K6PC-5, an activator of SphK1, or overexpression of SphK1 significantly attenuated the anti-proliferative and pro-apoptotic effects of hispidulin. In addition, hispidulin treatment dose-dependently activated ROS/JNK signaling and led to cell apoptosis. We further demonstrated in Caki-2 xenograft nude mice that injection of hispidulin (20, 40 mg·kg -1 ·d -1 , ip) dose-dependently suppressed tumor growth accompanied by decreased SphK1 activity and increased ceramide accumulation in tumor tissues. Our findings reveal a new explanation for the anti-tumor mechanisms of hispidulin, and suggest that SphK1 and ceramide may serve as potential therapeutic targets for the treatment of ccRCC.

Malignant mesothelioma (MM) is an aggressive malignancy, highly resistant to current medical and surgical therapies, whose tumor cells characteristically show a high level of aneuploidy and genomic instability. We tested our hypothesis that targeting chromosomal instability in MM would improve response to therapy. Thr/Tyr kinase (TTK)/monopolar spindle 1 kinase (Mps-1) is a kinase of the spindle assembly checkpoint that controls cell division and cell fate. CFI-402257 is a novel, selective inhibitor of Mps-1 with antineoplastic activity. We found that CFI-402257 suppresses MM growth. We found that Mps-1 is overexpressed in MM and that its expression correlates with poor patients' outcome. In vitro, CFI-402257-mediated inhibition of Mps-1 resulted in abrogation of the mitotic checkpoint, premature progression through mitosis, marked aneuploidy and mitotic catastrophe. In vivo, CFI-402257 reduced MM growth in an orthotopic, syngeneic model, when used as a single agent, and more so when used in combination with cisplatin+pemetrexed, the current standard of care. Our preclinical findings indicate that CFI-402257 is a promising novel therapeutic agent to improve the efficacy of the current chemotherapeutic regimens for MM patients.

Network pharmacology has become a powerful means of understanding the mechanisms underlying the action of Chinese herbs in cancer treatment. This study aims to validate the preventive effects and molecular mechanisms of a clinical prescription XIAOPI formula against breast cancer. In vivo breast cancer xenograft data showed that XIAOPI delayed breast cancer development and efficiently inhibited lung metastasis, accompanied by prolonged survival benefits and decreased cancer stem cell subpopulations. However, similar phenomenon were not observed in a cell model. The herb-ingredient-target network analysis further identified a total of 81 genes closely correlated with the breast cancer chemoprevention effects of XIAOPI. Cytokine array analysis further validated CXCL-1 as the key target of XIAOPI both in vitro and in vivo. Evaluation of the mechanism demonstrated that CXCL-1 administration significantly abrogated the metastatic inhibition effects of XIAOPI on breast cancer migration, invasion, stem cells subpopulations, epithelial-mesenchymal transition(EMT), or mammosphere formation abilities. Overall, our study provides experimental evidence and molecular mechanisms that may facilitate the safe and effective use of herbal medicine for the prevention of breast cancer growth or metastasis, and may lead to CXCL-1-based therapeutic strategies for mammary malignancies.

Low-molecular-weight Fucoidan (Oligo-Fucoidan) is a sulfated polysaccharide that has a variety of biological effects and has also been shown to have beneficial health effects. However, the molecular mechanisms underlying the therapeutic effects of Oligo-Fucoidan in patients with cancer remain unclear. Using human colorectal cancer HCT116 cells with (p53 +/+ ) or without (p53 -/- ) normal p53 expression, we found that Oligo-Fucoidan treatment reduces the occurrence of spontaneous DNA lesions. Etoposide induces double strand DNA breaks. Subsequent administration of Oligo-Fucoidan to etoposide-treated cells promotes p53 accumulation, p21 expression and significant decreases in ataxia-telangiectasia-mutated (ATM), checkpoint kinase 1 (Chk1) and γ-H2AX phosphorylation in p53 +/+ cells compared with p53 -/- cells. Similarly, co-administration of Oligo-Fucoidan with etoposide inhibits ATM, Chk1 and γ-H2AX phosphorylation, particularly in the presence of p53. Furthermore, Oligo-Fucoidan supplementation increases cancer cell death and attenuates the adverse effects induced by etoposide that decreases production of the pro-inflammatory cytokine IL-6 and chemokine CCL2/MCP-1. Importantly, Oligo-Fucoidan decreases the tumor-promoting M2 macrophages in microenvironment as well as collaborates with p53 and works in combination with etoposide to prevent HCT116 tumorigenicity. Our results first demonstrate that p53 enables Oligo-Fucoidan to effectively inhibit tumor progression, and Oligo-Fucoidan minimizes the side effects of chemotherapy and alters tumor microenvironment.