MFI2 upregulation promotes malignant progression through EGF/FAK signaling in oral cavity squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is the predominant histological type of the head and neck squamous cell carcinoma (HNSCC). By comparing the differentially expressed genes (DEGs) in OSCC-TCGA patients with copy number variations (CNVs) that we identify in OSCC-OncoScan dataset, we herein identified 37 dysregulated candidate genes. Among these potential candidate genes, 26 have been previously reported as dysregulated proteins or genes in HNSCC. Among 11 novel candidates, the overall survival analysis revealed that melanotransferrin (MFI2) is the most significant prognostic molecular in OSCC-TCGA patients. Another independent Taiwanese cohort confirmed that higher MFI2 transcript levels were significantly associated with poor prognosis. Mechanistically, we found that knockdown of MFI2 reduced cell viability, migration and invasion via modulating EGF/FAK signaling in OSCC cells. Collectively, our results support a mechanistic understanding of a novel role for MFI2 in promoting cell invasiveness in OSCC.

Radiotherapy enhances CXCR3highCD8+ T cell activation through inducing IFNγ-mediated CXCL10 and ICAM-1 expression in lung cancer cells.

Radiotherapy (RT) not only damages tumors but also induces interferon (IFN) expression in tumors. IFNs mediate PD-L1 to exhaust CD8+ T cells, but which also directly impact tumor cells and potentially activate anti-tumor immune surveillance. Little is known about the contradictory mechanism of IFNs in regulating CD8+ T-mediated anti-tumor activity in lung cancer. This study found that RT induced IFNs and CXCL9/10 expression in the RT-treated lung cancer cells. Specifically, RT- and IFNγ-pretreated A549 significantly activated CD8+ T cells, resulting in significant inhibition of A549 colony formation. RNAseq and consequent qPCR results revealed that IFNγ induced PD-L1, CXCL10, and ICAM-1, whereas PD-L1 knockdown activated CD8+ T cells, but ICAM-1 knockdown diminished CD8+ T cell activation. We further demonstrated that CXCR3 and CXCL10 decreased in the CD8+ T cells and nonCD8+ PBMCs, respectively, in the patients with lung cancer that expressed lower reactivation as co-cultured with A549 cells. In addition, inhibitors targeting CXCR3 and LFA-1 in CD8+ T cells significantly diminished CD8+ T cell activation and splenocytes-mediated anti-LL/2shPdl1. In conclusion, we validated that RT suppressed lung cancer and overexpress PD-L1, CXCL10, and ICAM-1, which exhibited different roles in regulating CD8+ T cell activity. We propose that CXCR3highCD8+ T cells stimulated by CXCL10 exhibit anti-tumor immunity, possibly by enhancing T cells-tumor cells adhesion through CXCL10/CXCR3-activated LFA-1-ICAM-1 interaction, but CXCR3lowCD8+ T cells with low CXCL10 in patients with lung cancer were exhausted by PD-L1 dominantly. Therefore, RT potentially activates CD8+ T cells by inducing IFNs-mediated CXCL10 and ICAM-1 expression in tumors to enhance CD8+ T-tumor adhesion and recognition. This study clarified the possible mechanisms of RT and IFNs in regulating CD8+ T cell activation in lung cancer.

Sorafenib suppresses radioresistance and synergizes radiotherapy-mediated CD8+ T cell activation to eradicate hepatocellular carcinoma.

Radiotherapy (RT) is applied to eradicate tumors in the clinic. However, hepatocellular carcinoma (HCC) exhibits resistance against RT. It is demonstrated that RT directly inhibits tumor growth but which induces type I interferons (IFNs) expression to phosphorylate STATs and increase STATs-downstream PD-L1 levels in the survival tumor cells. Since sorafenib is capable of suppressing STATs, we, therefore, hypothesize that sorafenib suppresses IFNs-mediated radioresistance and PD-L1 in the residual tumor cells and may synergistically enhance RT-mediated reactivation of CD8+ T immunological activity to eradicate HCC cells. We found that combined RT, sorafenib, and PBMCs significantly suppress the colony formation in the HCC cells, whereas CD8+ T cells expressed high granzyme B (GZMB) and perforin (PRF1) in co-cultured with RT-treated HCC cells. We demonstrated RT significantly inhibited HCC cell viability but induced IFNα and IL-6 expression in the RT-treated HCC cells, resulting in immune checkpoint PD-L1 and anti-apoptosis MCL1 and BCL2 overexpression in the non-RT HCC cells. We found that sorafenib decreased RT-PLC5 medium (RT-PLC5-m)-mediated cell growth by suppressing IFNα- and IL-6-mediated STAT1 and STAT3 phosphorylation. Sorafenib also reduced IFNα-mediated PD-L1 levels in HCC cells. Meanwhile, RT-PLC5-m reactivated CD8+ T cells and non-CD8+ PBMCs, resulting in high IFNγ and IL-2 levels in CD8+ T cells, and cytokines IFNα, IFNγ, IL-2, and IL-6 in non-CD8+ PBMCs. Particularly, CD8+ T cells expressed higher GZMB and PRF1 and non-CD8+ PBMCs expressed higher IFNα, IFNγ, IL-2, IL-6, CXCL9, and CXCL10 in co-cultured with RT-treated HCC cells compared to parental cells. Although we demonstrated that sorafenib slightly inhibited RT-mediated GZMB and PRF1 expression in CD8+ T cells, and cytokines levels in non-CD8+ PBMCs. Based on sorafenib significantly suppressed IFNα- and IL-6-mediated radioresistance and PD-L1 expression, we demonstrated that sorafenib synergized RT and immune surveillance for suppressing PLC5 cell viability in vitro. In conclusion, this study revealed that RT induced IFNα and IL-6 expression to phosphorylate STAT1 and STAT3 by autocrine and paracrine effect, leading to radioresistance and PD-L1 overexpression in HCC cells. Sorafenib not only suppressed IFNα- and IL-6-mediated PLC5 cell growth but also inhibited IFNα-mediated PD-L1 expression, synergistically enhancing RT-mediated CD8+ T cell reactivation against HCC cells.

Preclinical Therapeutic Assessment of a New Chemotherapeutics [Dichloro(4,4'-Bis(2,2,3,3-Tetrafluoropropoxy) Methyl)-2,2'-Bipryridine) Platinum] in an Orthotopic Patient-Derived Xenograft Model of Triple-Negative Breast Cancers.

Cisplatin is one of the most common therapeutics used in treatments of several types of cancers. To enhance cisplatin lipophilicity and reduce resistance and side effects, a polyfluorinated bipyridine-modified cisplatin analogue, dichloro[4,4'-bis(2,2,3,3-tetrafluoropropoxy)methyl)-2,2'-bipryridine] platinum (TFBPC), was synthesized and therapeutic assessments were performed. TFBPC displayed superior effects in inhibiting the proliferation of several cisplatin-resistant human cancer cell lines, including MDA-MB-231 breast cancers, COLO205 colon cancers and SK-OV-3 ovarian cancers. TFBPC bound to DNA and formed DNA crosslinks that resulted in DNA degradation, triggering the cell death program through the PARP/Bax/Bcl-2 apoptosis and LC3-related autophagy pathway. Moreover, TFBPC significantly inhibited tumor growth in both animal models which include a cell line-derived xenograft model (CDX) of cisplatin-resistant MDA-MB-231, and a patient-derived xenograft (PDX) model of triple-negative breast cancers (TNBCs). Furthermore, the biopsy specimen from TFBPC-treated xenografts revealed decreased expressions of P53, Ki-67 and PD-L1 coupled with higher expression of cleaved caspase 3, suggesting TFBPC treatment was effective and resulted in good prognostic indications. No significant pathological changes were observed in hematological and biochemistry tests in blood and histological examinations from the specimen of major organs. Therefore, TFBPC is a potential candidate for treatments of patients suffering from TNBCs as well as other cisplatin-resistant cancers.

Improving Tirapazamine (TPZ) to Target and Eradicate Hypoxia Tumors by Gold Nanoparticle Carriers.

Tumor hypoxia is a hallmark of solid tumors and emerged as the therapeutic target for cancer treatments, such as a prodrug Tirapazamine (TPZ) activated in hypoxia. To increase tumor accumulation, gold nanoparticles (GNPs) were selected to conjugate with TPZ. In this study, we successfully formulated and assessed the biochemical and therapeutic roles of the conjugated gold nanoparticles-Tirapazamine (GNPs-TPZ) on therapeutic assessments of MKN45-induced xenograft animal model. The results indicated that GNPs-TPZ was a potential nanomedicine for selectively targeting hypoxia tumors coupled with decreased side effects on healthy tissue or organs. TPZ significantly reduced cell viability of hypoxic gastric cancer MKN45 cells, but not in cells incubated in normoxia condition. For improving tumor targeting efficiency, furthermore, the GNPs drug carrier was conjugated to TPZ via biding mediator bovine serum albumin (BSA), and we demonstrated that this conjugated GNPs-TPZ retained the unique characteristics of hypoxic toxin and possessed the adequate feature of systemic bio-distributions in animals. GNPs-TPZ nanoparticles revealed their superior affinity to hypoxia tumors in the MKN45 xenograft. Moreover, GNPs-TPZ treatments did not significantly alter the biochemical parameters of blood samples acquired from animals. Taken together, TPZ, a prodrug activated by hypoxia, was conjugated with GNPs, whereas BSA severed as an excellent binding agent for preparing the conjugated GNPs-TPZ nanomedicines. We demonstrated that GNPs-TPZ enhanced tumor targeting, resulting in higher therapeutic efficacy compared to TPZ. We suggest that it may sever as an adjuvant treatment or combined therapy with other chemotherapeutics for the treatment of cancer patients in the future.

Characterization of Recurrent Relevant Genes Reveals a Novel Role of RPL36A in Radioresistant Oral Squamous Cell Carcinoma.

Radioresistance is one of the major factors that contributes to radiotherapy failure in oral cavity squamous cell carcinoma (OSCC). By comparing the prognostic values of 20,502 genes expressed in patients in The Cancer Genome Atlas (TCGA)-OSCC cohort with (n = 162) and without radiotherapy (n = 118), herein identified 297 genes positively correlated with poor disease-free survival in OSCC patients with radiotherapy as the potential radioresistance-associated genes. Among the potential radioresistance-associated genes, 36 genes were upregulated in cancerous tissues relative to normal tissues. The bioinformatics analysis revealed that 60S ribosomal protein L36a (RPL36A) was the most frequently detected gene involved in radioresistance-associated gene-mediated biological pathways. Then, two independent cohorts (n = 162 and n = 136) were assessed to confirm that higher RPL36A transcript levels were significantly associated with a poor prognosis only in OSCC patients with radiotherapy. Mechanistically, we found that knockdown of RPL36A increased radiosensitivity via sensitizing cells to DNA damage and promoted G2/M cell cycle arrest followed by augmenting the irradiation-induced apoptosis pathway in OSCC cells. Taken together, our study supports the use of large-scale genomic data for identifying specific radioresistance-associated genes and suggests a regulatory role for RPL36A in the development of radioresistance in OSCC.

Irradiation Mediates IFNα and CXCL9 Expression in Non-Small Cell Lung Cancer to Stimulate CD8+ T Cells Activity and Migration toward Tumors.

Irradiation-broken DNA fragments increase type I interferon and chemokines secretion in tumor cells. Since radiotherapy may augment tumor immunotherapy, we hypothesize that the chemokines increased by irradiation could recruit CD8+ T cells to suppress tumor proliferation. This study intended to unveil the secreted factors activating and recruiting CD8+ T cells in non-small-cell lung cancer (NSCLC). EGFR-positive A549 was selected and treated by X-irradiation (IR) to identify the overexpression of chemokines associated to CD8+ T cell cytotoxicity and recruitment. A transwell assay with Alexa 488-labeled CD8+ T cells was used to evaluate CD8+ T cell motility in vitro. A nuclear imaging platform by In111-labeled nivolumab was used to track CD8+ T cells homing to tumors in vivo. The activation markers GZMB, PRF-1, and IFNγ, migration marker CD183 (CXCR3), and inhibitory marker CD274 (PD-1), were measured and compared in CD8+ T cells with A549 co-cultured, chemokines treated, and patients with late-stage lung cancer. We found that IR not only suppressed A549 proliferation but also induced IFNα and CXCL9 expression (p < 0.05). IFNα majorly increased IFNγ levels in CD8+ T cells (p < 0.05) and synergistically with CXCL9 enhanced CD8+ T cell migration in vitro (p < 0.05). We found that CXCR3 and PD-1 were down-regulated and up-regulated, respectively, in the peripheral blood CD8+ T cells in patients with lung cancer (n = 4 vs. healthy n = 3, both p < 0.05), which exhibited reduction of cell motility (p < 0.05). The in vivo nuclear imaging data indicated highly CD8+ T cells migrated to A549-induced tumors. In addition, we demonstrated that healthy PBMCs significantly suppressed the parallel tumor growth (p < 0.05) and the radioresistant tumor growth in the tumor xenograft mice (p < 0.05), but PBMCs from patients with lung cancer had lost the anti-tumor capacity. We demonstrated that IR induced IFNα and CXCL9 expression in A549 cells, leading to CD8+ T cell migration. This study unveiled a potential mechanism for radiotherapy to activate and recruit CD8+ T cells to suppress lung tumors.

Irradiation Suppresses IFNγ-Mediated PD-L1 and MCL1 Expression in EGFR-Positive Lung Cancer to Augment CD8+ T Cells Cytotoxicity.

Tumor cells express immune checkpoints to exhaust CD8+ T cells. Irradiation damages tumor cells and augments tumor immunotherapy in clinical applications. However, the radiotherapy-mediated molecular mechanism affecting CD8+ T cell activity remains elusive. We aimed to uncover the mechanism of radiotherapy augmenting cytotoxic CD8+ T cells in non-small-cell lung cancer (NSCLC). EGFR-positive NSCLC cell lines were co-cultured with CD8+ T cells from healthy volunteers. Tumor cell viability and apoptosis were consequently measured. IFNγ was identified secreted by CD8+ T cells and PBMCs. Therefore, RNAseq was used to screen the IFNγ-mediated gene expression in A549 cells. The irradiation effect to IFNγ-mediated gene expression was investigated using qPCR and western blots. We found that the co-culture of tumor cells stimulated the increase of granzyme B and IFNγ in CD8+ T, but A549 exhibited resistance against CD8+ T cytotoxicity compared to HCC827. Irradiation inhibited A549 proliferation and enhanced apoptosis, augmenting PBMCs-mediated cytotoxicity against A549. We found that IFNγ simultaneously increased phosphorylation on STAT1 and STAT3 in EGFR-positive lung cancer, resulting in overexpression of PD-L1 (p < 0.05). In RNAseq analysis, MCL1 was identified and increased by the IFNγ-STAT3 axis (p < 0.05). We demonstrated that irradiation specifically inhibited phosphorylation on STAT1 and STAT3 in IFNγ-treated A549, resulting in reductions of PD-L1 and MCL1 (both p < 0.05). Moreover, knockdowns of STAT3 and MCL1 increased the PBMCs-mediated anti-A549 effect. This study demonstrated that A549 expressed MCL1 to resist CD8+ T cell-mediated tumor apoptosis. In addition, we found that irradiation suppressed IFNγ-mediated STAT3 phosphorylation and PD-L1 and MCL1 expression, revealing a potential mechanism of radiotherapy augmenting immune surveillance.

Inhibition of glutaminolysis in combination with other therapies to improve cancer treatment.

Targeting glutamine catabolism has been attracting more research attention on the development of successful cancer therapy. Catalytic enzymes such as glutaminase (GLS) in glutaminolysis, a series of biochemical reactions by which glutamine is converted to glutamate and then alpha-ketoglutarate, an intermediate of the tricarboxylic acid (TCA) cycle, can be targeted by small molecule inhibitors, some of which are undergoing early phase clinical trials and exhibiting promising safety profiles. However, resistance to glutaminolysis targeting treatments has been observed, necessitating the development of treatments to combat this resistance. One option is to use synergy drug combinations, which improve tumor chemotherapy's effectiveness and diminish drug resistance and side effects. This review will focus on studies involving the glutaminolysis pathway and diverse combination therapies with therapeutic implications.

Nicotine exhausts CD8+ T cells against tumor cells through increasing miR-629-5p to repress IL2RB-mediated granzyme B expression.

The mechanism exhausting CD8+ T cells is not completely clear against tumors. Literature has demonstrated that cigarette smoking disables the immunological activity, so we propose nicotine is able to exhaust CD8+ T cells. The CD8+ T cells from healthy volunteers with and without cigarette smoking and the capacity of CD8+ T cells against tumor cells were investigated. RNAseq was used to investigate the gene profiling expression in CD8+ T cells. Meanwhile, small RNAseq was also used to search novel microRNAs involved in the exhaustion of CD8+ T cells. The effect of nicotine exhausting CD8+ T cells was investigated in vitro and in the humanized tumor xenografts in vivo. We found that CD8+ T cells were able to reduce cell viability in lung cancer HCC827 and A549 cells, that secreted granzyme B, but CD8+ T cells from the healthy cigarette smokers lost anti-HCC827 effect. Moreover, nicotine suppressed the anti-HCC827 effect of CD8+ T cells. RNAseq revealed lower levels of IL2RB and GZMB in the exhausted CD8+ T cells. We identified that miR-629-5p was increased by nicotine, that targeted IL2RB. Transfection of miR-629-5p mimic reduced IL2RB and GZMB levels. We further validated that nicotine reduced granzyme B levels using a nuclear imaging technique, and demonstrated that nicotine exhausted peripheral blood mononuclear cells against HCC827 growth in the humanized tumor xenografts. This study demonstrated that nicotine exhausted CD8+ T cells against HCC827 cells through increasing miR-629-5p to suppress IL2RB.

Low molecular weight fucoidan inhibits hepatocarcinogenesis and nonalcoholic fatty liver disease in zebrafish via ASGR/STAT3/HNF4A signaling.

BACKGROUND: Hepatocellular carcinoma ranks fourth in cancer-related mortality currently lacks effective therapeutics. Fucoidan is sulfated polysaccharide that is mainly found in brown seaweeds. In this study, we investigated the effects and mechanisms of low molecular weight fucoidan (i.e. oligo-fucoidan [OF]) preventing hepatocarcinogenesis. METHODS: We used [HBx,src], [HBx,src,p53-/+ ], and [CD36] transgenic zebrafish liver cancer model treated with OF, and performed molecular and histopathological analysis. Transcriptomic and pathways analysis was performed. RESULTS: Decreased expression of lipogenic enzymes, fibrosis markers, and cell cycle/proliferation markers by OF in [HBx,src] and [HBx,src,p53-/+ ] transgenic fish. Liver fibrosis was decreased as revealed by Sirius Red staining, and the liver cancer formation was eventually reduced by feeding OF. OF was also found to be capable of reducing lipid accumulation and cancer formation in non-B non-C Hepatocellular carcinoma (HCC) model in CD36 transgenic zebrafish. Whole-genome expression analysis showed that 661 genes were up-regulated, and 451 genes were downregulated by feeding OF. Upregulated genes were mostly found in protein transporter activity, and downregulated genes were enriched with response to extracellular stimulus and metal binding in gene ontology analysis. The driver gene was HNF4A revealed by NetworkAnalyst from OF differential regulated genes at various insults. OF is able to bind the asialoglycoprotein receptor (ASGR) in hepatoma cells, and increased the phosphorylation of signal transducer and activator of transcription 3 (STAT3) in both hepatoma cells and [HBx,src,p53-/+ ] transgenic fish liver cancer model. Using chromatin-immunoprecipitation, we found pSTAT3 could associate with the P1 promoter of HNF4A. Knockdown of either ASGR or HNF4A reversed OF mediated anti-cancer cell proliferation. CONCLUSIONS: Taken together, we provide evidence that OF exhibits the anti-HCC, anti-steatosis, and anti-fibrosis effect for liver in zebrafish models, and the anti-cancer potential of OF attributed to the binding to ASGR and activation of STAT3/HNF4A signaling. OF might be potentially valuable for the management of HCC.

STAT3 Mediated miR-30a-5p Inhibition Enhances Proliferation and Inhibits Apoptosis in Colorectal Cancer Cells.

Signal transducer and activator of transcription 3 (STAT3), a transcriptional factor involved in tumorigenesis and cancer stemness formation, contributes to drug resistance in cancer therapies. STAT3 not only mediates gene transcription but also participates in microRNA suppression. This study identified a STAT3-downstream micro RNA (miRNA) involved in drug resistance against regorafenib in colorectal cancer stem-like tumorspheres. Small RNAseq was used to investigate differential microRNAs in colorectal cancer cell-derived tumorspheres and in a STAT3-knockdown strain. The miRNA-mediated genes were identified by comparing RNAseq data with gene targets predicted using TargetScan. Assays for detecting cell viability and apoptosis were used to validate findings. The formation of colorectal cancer stem-like tumorspheres was inhibited by BBI608, a STAT3 inhibitor, but not by regorafenib. Additional investigations for microRNA expression demonstrated an increase in 10 miRNAs and a decrease in 13 miRNAs in HT29-derived tumorspheres. A comparison of small RNAseq results between tumorspheres and HT29shSTAT3 cells revealed the presence of four STAT3-mediated miRNAs in HT29-derived tumorspheres: hsa-miR-215-5p, hsa-miR-4521, and hsa-miR-215-3p were upregulated, whereas miR-30a-5p was downregulated. Furthermore, hsa-miR-4521 was associated with poor overall survival probability, and miR-30a-5p was associated with better overall survival probability in patients with rectum cancer. Comparisons of RNAseq findings between HCT116- and HT29-derived tumorspheres revealed that HSPA5 were mediated by the STAT3-miR-30a-5p axis, which is overexpressed in colorectal tumorspheres associating to anti-apoptosis. In addition, the transfection of miR-30a-5p and inhibition of HSPA5 by HA15 significantly reduced cell viability and increased apoptosis in HT29 cells. In conclusion, a STAT3-miR-30a-5p-HSPA5 axis was observed against regorafenib-mediated apoptosis in colorectal cancer tumorspheres. The expression of miR-30a-5p was repressed by STAT3; in addition, HSPA5 was identified as the target gene of miR-30a-5p and contributed to both tumorsphere formation and anti-apoptosis.

Discovery of Driver Genes in Colorectal HT29-derived Cancer Stem-Like Tumorspheres.

Cancer stem cells play a vital role against clinical therapies, contributing to tumor relapse. There are many oncogenes involved in tumorigenesis and the initiation of cancer stemness properties. Since gene expression in the formation of colorectal cancer-derived tumorspheres is unclear, it takes time to discover the mechanisms working on one gene at a time. This study demonstrates a method to quickly discover the driver genes involved in the survival of the colorectal cancer stem-like cells in vitro. Colorectal HT29 cancer cells that express the LGR5 when cultured as spheroids and accompany an increase CD133 stemness markers were selected and used in this study. The protocol presented is used to perform RNAseq with available bioinformatics to quickly uncover the overexpressed driver genes in the formation of colorectal HT29-derived stem-like tumorspheres. The methodology can quickly screen and discover potential driver genes in other disease models.

Low Molecular Weight Fucoidan Prevents Radiation-Induced Fibrosis and Secondary Tumors in a Zebrafish Model.

Radiotherapy often causes unwanted side effects such as radiation-induced fibrosis and second malignancies. Fucoidan, a sulfated polysaccharide extracted from brown seaweed, has many biological effects including anti-inflammation and anti-tumor. In the present study, we investigated the radioprotective effect of Oligo-Fucoidan (OF) using a zebrafish animal model. Adult zebrafish of wild-type and transgenic fish with hepatocellular carcinoma were orally fed with Oligo-Fucoidan before irradiation. Quantitative PCR, Sirius red stain, hematoxylin, and eosin stain were used for molecular and pathological analysis. Whole genomic microarrays were used to discover the global program of gene expression after Oligo-Fucoidan treatment and identified distinct classes of up- and downregulated genes/pathways during this process. Using Oligo-Fucoidan oral gavage in adult wild-type zebrafish, we found Oligo-Fucoidan pretreatment decreased irradiation-induced fibrosis in hepatocyte. Using hepatitis B virus X antigen (HBx), Src and HBx, Src, p53-/+ transgenic zebrafish liver cancer model, we found that Oligo-Fucoidan pretreatment before irradiation could lower the expression of lipogenic factors and enzymes, fibrosis, and cell cycle/proliferation markers, which eventually reduced formation of liver cancer compared to irradiation alone. Gene ontology analysis revealed that Oligo-Fucoidan pretreatment increased the expression of genes involved in oxidoreductase activity in zebrafish irradiation. Oligo-Fucoidan also decreased the expression of genes involved in transferase activity in wild-type fish without irradiation (WT), nuclear outer membrane-endoplasmic reticulum membrane network, and non-homologous end-joining (NHEJ) in hepatocellular carcinoma (HCC) transgenic fish. Rescue of those genes can prevent liver cancer formation. Conclusions: Our results provide evidence for the ability of Oligo-Fucoidan to prevent radiation-induced fibrosis and second malignancies in zebrafish.

Transcriptomically Revealed Oligo-Fucoidan Enhances the Immune System and Protects Hepatocytes via the ASGPR/STAT3/HNF4A Axis.

Oligo-fucoidan, a sulfated polysaccharide extracted from brown seaweed, exhibits anti-inflammatory and anti-tumor effects. However, the knowledge concerning the detailed mechanism of oligo-fucoidan on liver cells is obscure. In this study, we investigate the effect of oligo-fucoidan in normal hepatocytes by transcriptomic analysis. Using an oligo-fucoidan oral gavage in wild-type adult zebrafish, we find that oligo-fucoidan pretreatment enhances the immune system and anti-viral genes in hepatocytes. Oligo-fucoidan pretreatment also decreases the expression of lipogenic enzymes and liver fibrosis genes. Using pathway analysis, we identify hepatocyte nuclear factor 4 alpha (HNF4A) to be the potential driver gene. We further investigate whether hepatocyte nuclear factor 4 alpha (HNF4A) could be induced by oligo-fucoidan and the underlying mechanism. Therefore, a normal hepatocyte clone 9 cell as an in vitro model was used. We demonstrate that oligo-fucoidan increases cell viability, Cyp3a4 activity, and Hnf4a expression in clone 9 cells. We further demonstrate that oligo-fucoidan might bind to asialoglycoprotein receptors (ASGPR) in normal hepatocytes through both in vitro and in vivo competition assays. This binding, consequently activating the signal transducer and activator of transcription 3 (STAT3), increases the expression of the P1 isoform of HNF4A. According to our data, we suggest that oligo-fucoidan not only enhances the gene expression associated with anti-viral ability and immunity, but also increases P1-HNF4A levels through ASGPR/STAT3 axis, resulting in protecting hepatocytes.

STAT3 induces G9a to exacerbate HER3 expression for the survival of epidermal growth factor receptor-tyrosine kinase inhibitors in lung cancers.

BACKGROUND: HER3 mediates drug resistance against epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs), resulting in tumor relapse in lung cancers. Previously, we demonstrated that EGFR induces HER3 overexpression, which facilitates the formation of cancer stem-like tumorspheres. However, the cellular mechanism through which EGFR regulates HER3 expression remains unclear. We hypothesized that EGFR downstream of STAT3 participates in HER3 expression because STAT3 contributes to cancer stemness and survival of EGFR-TKI resistant cancers. METHODS: First, RNAseq was used to uncover potential genes involved in the formation of lung cancer HCC827-derived stem-like tumorspheres. EGFR-positive lung cancer cell lines, including HCC827, A549, and H1975, were individually treated with a panel containing 172 therapeutic agents targeting stem cell-associated genes to search for potential agents that could be applied against EGFR-positive lung cancers. In addition, gene knockdown and RNAseq were used to investigate molecular mechanisms through which STAT3 regulates tumor progression and the survival in lung cancer. RESULTS: BBI608, a STAT3 inhibitor, was a potential therapeutic agent that reduced the cell viability of EGFR-positive lung cancer cell lines. Notably, the inhibitory effects of BBI608 were similar with those associated with YM155, an ILF3 inhibitor. Both compounds reduced G9a-mediated HER3 expression. We also demonstrated that STAT3 upregulated G9a to silence miR-145-5p, which exacerbated HER3 expression in this study. CONCLUSIONS: The present study revealed that BBI608 could eradicate EGFR-positive lung cancers and demonstrated that STAT3 enhanced the expression of HER3 through miR-145-5p repression by G9a, indicating that STAT3 is a reliable therapeutic target against EGFR-TKI-resistant lung cancers.

STAT3 exacerbates survival of cancer stem-like tumorspheres in EGFR-positive colorectal cancers: RNAseq analysis and therapeutic screening.

BACKGROUND: Cancer stem cells are capable of undergoing cell division after surviving cancer therapies, leading to tumor progression and recurrence. Inhibitory agents against cancer stem cells may be therapeutically used for efficiently eradicating tumors. Therefore, the aim of this study was to identify the relevant driver genes that maintain cancer stemness in epidermal growth factor receptor (EGFR)-positive colorectal cancer (CRC) cells and to discover effective therapeutic agents against these genes. METHODS: In this study, EGFR-positive cancer stem-like cells (CSLCs) derived from HCT116 and HT29 cells were used as study models for in vitro inductions. To identify the differential genes that maintain CSLCs, RNAseq analysis was conducted followed by bioinformatics analysis. Moreover, a panel containing 172 therapeutic agents targeting the various pathways of stem cells was used to identify effective therapeutics against CSLCs. RESULTS: RNAseq analysis revealed that 654 and 840 genes were significantly upregulated and downregulated, respectively, in the HCT116 CSLCs. Among these genes, notably, platelet-derived growth factor A (PDGFA) and signal transducer and activator of transcription 3 (STAT3) were relevant according to the cancer pathway analyzed using NetworkAnalyst. Furthermore, therapeutic screening revealed that the agents targeting STAT3 and Wnt signaling pathways were efficient in reducing the cell viabilities of both HCT116 and HT29 cells. Consequently, we discovered that STAT3 inhibition using homoharringtonine and STAT3 knockdown significantly reduced the formation and survival of HT29-derived tumorspheres. We also observed that STAT3 phosphorylation was regulated by epidermal growth factor (EGF) to induce PDGFA and Wnt signaling cascades. CONCLUSIONS: We identified the potential genes involved in tumorsphere formation and survival in selective EGFR-positive CRCs. The results reveal that the EGF-STAT3 signaling pathway promotes and maintains CRC stemness. In addition, a crosstalk between STAT3 and Wnt activates the Wnt/ß-catenin signaling pathway, which is also responsible for cancer stemness. Thus, STAT3 is a putative therapeutic target for CRC treatment.

Epidermal growth factor induces STAT1 expression to exacerbate the IFNr-mediated PD-L1 axis in epidermal growth factor receptor-positive cancers.

The epidermal growth factor (EGF) receptor (EGFR) overexpressed in many cancers, including lung and head and neck cancers, and is involved in cancer cell progression and survival. PD-L1, increases in tumor cells to evade and inhibit CD8+ T cells, is a clinical immunotherapeutic target. This study investigated the molecular mechanism of EGF on regulating PD-L1 in EGFR-positive cancers and determined potential agents to reduce PD-L1 expression. RNA sequencing (RNAseq) and bioinformatics analysis were performed to determine potential driver genes that regulate PD-L1 in tumor cells-derived tumorspheres which mimicking cancer stem cells. Then, the specific inhibitors targeting EGFR were applied to reduce the expression of PD-L1 in vitro and in vivo. We validated that EGF could induce PD-L1 expression in the selected EGFR-positive cancers. RNAseq results revealed that STAT1 increased as a driver gene in KOSC-3-derived tumorspheres; these data were analyzed using PANTHER followed by NetworkAnalyst. The blockade of EGFR by afatinib resulted in decreased STAT1 and IRF-1 levels, both are transcriptional factors of PD-L1, and disabled the IFNr-STAT1-mediated PD-L1 axis in vitro and in vivo. Moreover, STAT1 knockdown significantly reduced EGF-mediated PD-L1 expression, and ruxolitinib, a JAK1/JAK2 inhibitor, significantly inhibited STAT1 phosphorylation to reduce the IFNr-mediated PD-L1 axis. These results indicate that EGF exacerbates PD-L1 by increasing the protein levels of STAT1 to enforce the IFNr-JAK1/2-mediated signaling axis in selected EGFR-positive cancers. The inhibition of EGFR by afatinib significantly reduced PD-L1 and may be a potential strategy for enhancing immunotherapeutic efficacy.

Anti-angiogenic treatment (Bevacizumab) improves the responsiveness of photodynamic therapy in colorectal cancer.

Photodynamic therapy (PDT) is a treatment utilizing the combined action of photosensitizers and light for the treatment of various cancers. The mechanisms for tumor destruction after PDT include direct tumor cell kill by singlet oxygen species (OS), indirect cell kill via vascular damage, and an elicited immune response. However, it has been reported that many cellular activators, including vascular endothelial growth factor (VEGF), are produced by tumor cells after PDT. In this study, we demonstrate that meta-tetra(hydroxyphenyl) chlorin (mTHPC)-based photodynamic therapy combined with bevacizumab (Avastin™), an anti-VEGF neutralizing monoclonal antibody that blocks the binding of VEGF to its receptor, can enhance the effectiveness of each treatment modality. We evaluated the efficacy of bevacizumab-based anti-angiogenesis in combination with PDT as well as the resulting VEGF levels and microvessel density (MVD) in a mouse model of human colon cancer. Enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry (IHC) were performed to assess VEGF concentrations and microvessel density in the various treatment groups, and confocal imaging and high performance liquid chromatography (HPLC) analyses were used to measure the distribution and concentration of mTHPC in tumors. Our results demonstrate that combination of PDT followed by bevacizumab significantly elicits a greater tumor response whereas bevacizumab treatment prior to PDT led to a reduced tumor response. Immunostaining and ELISA analyses revealed a lower expression of VEGF in tumors treated with combination therapy of PDT followed by bevacizumab. However, bevacizumab treatment decreased the accumulation of mTHPC in tumors 24 h after administration, which complemented the results of decreased anti-tumor efficacy of bevacizumab followed by PDT.

EGFR-mediated interleukin enhancer-binding factor 3 contributes to formation and survival of cancer stem-like tumorspheres as a therapeutic target against EGFR-positive non-small cell lung cancer.

OBJECTIVES: YM155, an inhibitor of interleukin enhancer-binding factor 3 (ILF3), significantly suppresses cancer stemness property, implying that ILF3 contributes to cell survival of cancer stem cells. However, the molecular function of ILF3 inhibiting cancer stemness remains unclear. This study aimed to uncover the potential function of ILF3 involving in cell survival of epidermal growth factor receptor (EGFR)-positive lung stem-like cancer, and to investigate the potential role to improve the efficacy of anti-EGFR therapeutics. MATERIALS AND METHODS: The association of EGFR and ILF3 in expression and regulations was first investigated in this study. Lung cancer A549 cells with deprivation of ILF3 were created by the gene-knockdown method and then RNAseq was applied to identify the putative genes regulated by ILF3. Meanwhile, HCC827- and A549-derived cancer stem-like cells were used to investigate the role of ILF3 in the formation of cancer stem-like tumorspheres. RESULTS: We found that EGFR induced ILF3 expression, and YM155 reduced EGFR expression. The knockdown of ILF3 reduced not only EGFR expression in mRNA and protein levels, but also cell proliferation in vitro and in vivo, demonstrating that ILF3 may play an important role in contributing to cancer cell survival. Moreover, the knockdown and inhibition of ILF3 by shRNA and YM155, respectively, reduced the formation and survival of HCC827- and A549-derived tumorspheres through inhibiting ErbB3 (HER3) expression, and synergized the therapeutic efficacy of afatinib, a tyrosine kinase inhibitor, against EGFR-positive A549 lung cells. CONCLUSION: This study demonstrated that ILF3 plays an oncogenic like role in maintaining the EGFR-mediated cellular pathway, and can be a therapeutic target to improve the therapeutic efficacy of afatinib. Our results suggested that YM155, an ILF3 inhibitor, has the potential for utilization in cancer therapy against EGFR-positive lung cancers.