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Abstract Chronic hepatitis B (CHB) leads to liver inflammation and dysfunction, resulting in liver fibrosis and cancer. Nucleos(t)ide analogues (NAs), inhibitors of hepatitis B virus (HBV), specifically suppress HBV replication. We proposed that immune modulation benefits seroconversion by HBsAg loss. However, activation of T lymphocytes also deteriorates hepatic inflammation. Therefore, we intended to investigate the T cell status and its relationship with hepatic functions in CHB patients treated with NAs. Serum markers, including liver function markers AST, ALT, and HBV-infected markers HBV DNA, HBsAg, HBeAg, and HBsAb were measured in the clinical routine. The T cell levels and markers, including CD69, CD107a, CXCR3, and PD-1 were investigated using flow cytometry. Meanwhile, IFNγ, IL-2, and CXCL10 as immune activation markers in the PBMCs were investigated using qPCR. To validate the effects of NAs on T cell status, qPCR and flow cytometry were used to investigate the gene expression in the HepG2 and PLC5 cells treated with NAs, and in the healthy PBMCs treated with the cell-cultured supernatant. We found that NAs significantly suppressed HBV DNA and reduced AST and ALT levels in the CHB patients. Meanwhile, AST and ALT were both positively correlated with activation marker CD107a in CD8+ T cells. In addition, we found that the CHB patients with seroconversion exhibited a higher CD4/CD8 ratio (p

Abstract 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.

Abstract 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/β-catenin signaling pathway, which is also responsible for cancer stemness. Thus, STAT3 is a putative therapeutic target for CRC treatment.

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.

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.

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.

Cancer stem cell survival is the leading factor for tumor recurrence after tumor-suppressive treatments. Therefore, specific and efficient inhibitors of cancer stemness must be discovered for reducing tumor recurrence. YM155 has been indicated to significantly reduce stemness-derived tumorsphere formation. However, the pharmaceutical mechanism of YM155 against cancer stemness is unclear. This study investigated the potential mechanism of YM155 against cancer stemness in lung cancer. Tumorspheres derived from epidermal growth factor receptor (EGFR)-mutant HCC827 and EGFR wild-type A549 cells expressing higher cancer stemness markers (CD133, Oct4, and Nanog) were used as cancer stemness models. We observed that EGFR autophosphorylation (Y1068) was higher in HCC827- and A549-derived tumorspheres than in parental cells; this autophosphorylation induced tumorsphere formation by activating G9a-mediated stemness. Notably, YM155 inhibited tumorsphere formation by blocking the autophosphorylation of EGFR and the EGFR-G9a-mediated stemness pathway. The chemical and genetic inhibition of EGFR and G9a revealed the significant role of the EGFR-G9a pathway in maintaining the cancer stemness property. In conclusion, this study not only revealed that EGFR could trigger tumorsphere formation by elevating G9a-mediated stemness but also demonstrated that YM155 could inhibit this formation by simultaneously blocking EGFR autophosphorylation and G9a activity, thus acting as a potent agent against lung cancer stemness.

Background: Hypoxia in tumor niche is one of important factors to start regeneration of blood vessels, leading to increase survival, proliferation, and invasion in cancer cells. Under hypoxia microenvironment, furthermore, steadily increased hypoxia-inducible factor-1α (HIF-1α) is observed, and can increase vascular endothelial growth factor (VEGF) expression and promote angiogenesis. Zinc protoporphyrin (ZnPP), a heme oxygenase-1 (HO-1) inhibitor, is potential to inhibit tumor proliferation and progression. However, the mechanism of ZnPP in inhibition of tumor is not completely clear. We hypothesize that ZnPP may modulate HIF-1α through inhibiting HO-1, and then inhibit angiogenesis and tumor progression. This study aimed to dissect the mechanism of ZnPP in tumor suppression. Results: We observed the amount of VEGF was increased in the sera of the colorectal cancer (CRC) patients (n = 34, p < 0.05). Furthermore, increased VEGF expression was also measured in colorectal cancer cells, HCT-15, culturing under mimicking hypoxic condition. It suggested that hypoxia induced VEGF production from cancer cells. VEGF production was significantly reduced from HCT-15 cells after exposure to HIF-1α inhibitor KC7F2, suggesting that HIF-1α regulated VEGF production. Moreover, we observed that the HO-1inhibitor ZnPP inhibited the expressions of HIF-1α and VEGF coupled with cell proliferations of HCT-15 cells, suggesting that ZnPP blocked HIF-1α expression, and then inhibited the consequent VEGF production. In the xenograft model, we also observed that the animals exposed to ZnPP displayed much smaller tumor nodules and less degree of angiogenesis with decreased expression of the angiogenesis marker, αvβ3 integrin, compared to that in normal control. Conclusions: This study demonstrated that VEGF level in serum was elevated in the patients with CRC. The HO-1 inhibitor, ZnPP, possessed the properties of anti-tumor agent by decreasing HIF-1α levels, blocking VEGF production, impairing tumor angiogenesis, and inhibiting tumor growth. [ABSTRACT FROM AUTHOR]

Background: The gold standard of assessing liver fibrosis is liver biopsy, which is invasive and not without risk. Therefore, searching for noninvasive serologic biomarkers for liver fibrosis is an importantly clinical issue. Methods: A total of 16 healthy volunteers and 45 patients with chronic hepatitis C virus (HCV) were enrolled (F0: n = 16, F1: n = 7, F2: n = 17, F3: n = 8 and F4: n = 13, according to the METAVIR classification). Three serum samples of each fibrotic stage were analyzed by two-dimension difference gel electrophoresis (2D-DIGE). The differential proteins were identified by the cooperation of MALDI-TOF/TOF and MASCOT; then western blotting and Bio-Plex Suspension Array were used to quantify the protein levels. Results: Three prominent candidate biomarkers were identified: alpha 2 macroglobulin (A2M) is up regulated; vitamin D binding protein (VDBP) and apolipoprotein AI (ApoAI) are down regulated. The serum concentration of A2M was significantly different among normal, mild (F1/F2) and advanced fibrosis (F3/F4) (p < 0.01). The protein levels of VDBP and ApoAI were significantly higher in normal/mild fibrosis, when compared to those in advanced fibrosis (both p < 0.01). Conclusions: This study not only reveals three putative biomarkers of liver fibrosis (A2M, VDBP and ApoAI) but also proves the differential expressions of those markers in different stages of fibrosis. We expect that combination of these novel biomarkers could be applied clinically to predict the stage of liver fibrosis without the need of liver biopsy. [ABSTRACT FROM AUTHOR]