miR-26b-5p helps in EpCAM+cancer stem cells maintenance via HSC71/HSPA8 and augments malignant features in HCC.

BACKGROUND: Targeting cancer stem cells (CSCs) in hepatocellular carcinoma (HCC) is difficult because of their similarities with normal stem cells (NSCs). EpCAM can identify CSCs from EpCAM+AFP+HCC cases, but is also expressed on NSCs. We aimed to distinguish the two using integrated protein, mRNA and miRNA profiling. METHODS: iTRAQ based protein profiling and Next Generation Sequencing (NGS) was performed on EpCAM+/EpCAM- cells isolated from HCC (Ep+CSC, Ep- HCC) and EpCAM+ cells from non-cancerous/non-cirrhotic control liver tissues (Ep+NSC). Validations were done using qRT-PCR, flowcytometry and western blotting followed by in vitro and in vivo functional studies. RESULTS: 11 proteins were overexpressed (>3 fold) in Ep+CSCs compared to Ep- HCC and Ep+NSC cells. However, RNA-sequencing confirmed the Ep+CSC specific up-regulation of only HSPA8, HNRNPC, MPST and GAPDH mRNAs among these. Database search combined with miRNA profiling revealed Ep+ CSC specific down-regulation of 29 miRNAs targeting these four genes. Of these, only miR-26b-5p was found to target both HSPA8 and EpCAM. Validation of HSPA8 overexpression and miR-26b-5p down-regulation followed by linear regression analysis established a negative correlation between the two. Functional studies demonstrated that reduced miR-26b-5p expression increased the spheroid formation, migration, invasion and tumourigenicity of Ep+ CSCs. Furthermore, anti-miR-26b-5p increased the number of Ep+ CSCs with a concomitant overexpression of stemness genes and reduction of proapoptotic protein BBC3, which is a known substrate of HSPA8. CONCLUSION: miR-26b-5p imparts metastatic properties and helps in maintenance of Ep+ CSCs via HSPA8. Thus, miR-26b-5p and HSPA8 could serve as molecular targets for selectively eliminating the Ep+ CSC population in human HCCs.

Dichotomy of Notch signalling in regulating tumour immune surveillance.

Notch signalling is an evolutionarily conserved multifaceted pathway that controls diverse cellular processes. Its role in regulating development and tissue homeostasis is well established. Aberrant activation of the Notch pathway has been implicated in the initiation and progression of many types of cancers. However, although in some cancers Notch signalling acts as a tumour-promoter, in others it is reported to suppress tumour growth and progression. Accumulating evidence suggests the involvement of both the innate and adaptive immune system in the development of various tumours. Currently, extensive studies on investigating the effects of Notch signalling in tumour immune surveillance are being carried out. Interestingly, recent literature shows how the changing expression of Notch genes in different T cell subsets like CD4 and CD8 helps in controlling anti-tumour immune responses. In this review, we discuss in depth the roles of Notch signalling molecules and different immune cells in the context of the tumour microenvironment. We also outline how current knowledge can be exploited to develop novel therapies in order to control the propagation of cancer stem cells.

miRNA signatures can predict acute liver failure in hepatitis E infected pregnant females.

BACKGROUND: Acute viral hepatitis E (AVH-E) can often result in acute liver failure (ALF) during pregnancy. microRNAs serve as mediators in drug induced liver failure. We investigated their role as a biomarker in predicting ALF due to HEV (ALF-E). METHODS: We performed next generation sequencing and subsequent validation studies in PBMCs of pregnant (P) self limiting AVH-E, ALF due to HEV (ALF-E) and compared with AVH-E in non-pregnant (NP) females and healthy controls. FINDINGS: Eleven microRNAs were significantly expressed in response to HEV infection; importantly, miR- 431, 654, 1468 and 4435, were distinctly expressed in pregnant self-limiting AVH-E and healthy females (p = 0.0005), but not in ALF-E. Sixteen exclusive microRNAs differentiated ALF-E from self limiting AVH-E in pregnant females. miR-450b which affects cellular proliferation and metabolic processes through RNF20 and SECB was predominanlty upregulated and correlated with poor outcome (ROC 0.958, p = 0.001). INTERPRETATION: Our results reveal that a specific microRNA profile can predict fatality in ALF-E in pregnancy. These microRNAs could be exploited as prognostic biomarkers and help in the development of new therapeutic interventions.

EpCAM+ Liver Cancer Stem-Like Cells Exhibiting Autocrine Wnt Signaling Potentially Originate in Cirrhotic Patients.

Hepatocellular carcinoma (HCC) is believed to originate from cancer stem cells (CSCs). While epithelial cell adhesion molecule (EpCAM) is a marker of normal hepatic stem cells (HSCs), EpCAM+ cells from HCC behave like CSCs. Since HCC mostly develops on a cirrhotic background, we sought to determine whether CSC-like EpCAM+ cells exist in patients with advanced cirrhosis. Both flow cytometry and immunohistochemistry showed that frequency of EpCAM+ cells in advanced cirrhosis was increased as compared to control. To determine whether increased EpCAM population in advanced cirrhosis harbors any CSC-like cells, we compared molecular and functional features of EpCAM+ cells from advanced cirrhosis (Ep+CIR; n = 20) with EpCAM+ cells from both HCC (Ep+HCC; n = 20) and noncancerous/noncirrhotic (control) (Ep+NSC; n = 7) liver tissues. Ep+CIRs displayed similarity with Ep+HCC cells including upregulated expression of stemness and Notch pathway genes, enhanced self-renewal in serial spheroid assay and generation of subcutaneous tumors in nonobese diabetic/severe combined immunodeficiency mice. Moreover, transcriptome and miRNome of Ep+CIRs appeared closer to that of Ep+HCC cells than Ep+NSCs. Interestingly, more than 50% micro RNAs (miRNAs) and transcripts specifically expressed in Ep+HCCs were also expressed in Ep+CIRs. However, none of Ep+NSC specific miRNAs and only 7% Ep+NSC specific transcripts were expressed in Ep+CIRs. Further, according to gene expression and in vitro Wnt inhibition analysis, autocrine Wnt signaling appeared to be a distinct feature of Ep+CIR and Ep+HCC cells, which was absent from Ep+NSCs. EpCAM+ cells in advanced cirrhosis possibly include a population of CSC-like cells which can be explored for early diagnosis of HCC development. Stem Cells Translational Medicine 2017;6:807-818.

CD4+CD25+CD127(low) Regulatory T Cells Play Predominant Anti-Tumor Suppressive Role in Hepatitis B Virus-Associated Hepatocellular Carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is the second leading cause of cancer death worldwide and hepatitis B is one of the commonest causes. T regulatory cells (Tregs) are strong immunomodulators and are likely to play a major role in HCC development. HBV infection is reported to induce expansion of Tregs. We investigated the CD4+CD25+CD127(-ve)FoxP3+ Tregs in HBV-related HCC as compared to non-HBV-HCC. PATIENTS AND METHODS: Whole blood immunophenotyping was analyzed by multicolor flow cytometry in patients with HBV-related HCC (HBV-HCC, n = 17), non-HBV-HCC (n = 22; NASH = 16, alcohol-related = 6), and chronic hepatitis B infection (CHBV; n = 10). Tregs functionality was checked by in vitro suppression assays using CD4+ CD25+ CD127(low) Tregs. Levels of serum alpha-fetoprotein (AFP), expression of FoxP3, IL-10, PD1, TGF-ß, and Notch in Tregs, and liver explants were analyzed by flow cytometry, immunohistochemistry, and quantitative RT-PCR. RESULTS: CD4+CD25+(hi) and Foxp3 expression in CD4+CD25+(hi)CD127(low) was significantly increased (P = 0.04, P = 0.007) in HBVHCC compared to non-HBVHCC and CHBV patients. HBVHCC also showed high IL-10 and TGF-ß secreting CD4 + CD25 + (hi)Tregs. The PD1 expression in CD4 + CD25+(hi) was significantly decreased in the HBVHCC than non-HBVHCC. In HBVHCC, AFP levels were significantly high (median 941, range 2-727940) than non-HBVHCC (median 13.5, range 2-18,900). In HBVHCC, patients with high AFP (range; 3982-727940 ng/ml) showed positive correlation with Foxp3 expression in CD4+CD25+(hi) CD127(low) (r = 0.857, P = 0.014). Reduced PD1 expression in HBVHCC also had negative correlation with FOXP3 in CD4+CD25+(hi) CD127(low) (r = -0.78, P = 0.04). However, AFP levels in non-HBVHCC showed negative correlation with (R = -0.67, P = 0.005) with CD4+CD25+(hi) Tregs. CONCLUSION: Our results demonstrate that CD4+ CD25+(hi) Tregs from HBVHCC patients have decreased expression of PD1, resulting in higher IL-10 and TGF-ß secretion. Increased suppressive ability of Tregs in HBV-related HCC confers increased anti-tumor suppressive response than in non-HBV-HCC. Modulation of Tregs and PD1 may serve as useful therapeutic targets.

Improving the anticancer activity of curcumin using nanocurcumin dispersion in water.

Curcumin is a highly potent, nontoxic bioactive agent found in turmeric and is known to have significant anticancer properties against different types of cancer cells. The major disadvantage associated with the use of curcumin, however, is its low systemic bioavailability due to its poor aqueous solubility. The focus of the present study was to generate nanoparticles of curcumin with improved aqueous phase solubility, and to investigate their efficacy in treating cancer cells. Curcumin nanoparticles having particle size in the range 2-40 nm and aqueous solubility of up to a maximum of 3 mg/mL were prepared. Evaluation of anticancer properties of curcumin nanodispersion was carried out in 3 different cancer cell lines: lung (A549), liver (HepG2), and skin (A431). The results demonstrated that under aqueous conditions curcumin nanoparticles exhibited similar or a much stronger antiproliferative effect on the cancer cells compared to normal curcumin in DMSO. Our results lead way toward unharnessed potential of curcumin in the form of its nanoparticles as an adjuvant therapy for clinical application in treating various cancers.

From cirrhosis to hepatocellular carcinoma: new molecular insights on inflammation and cellular senescence.

Sequential progression from chronic liver disease to fibrosis and to cirrhosis culminates in neoplasia in hepatocellular carcinoma (HCC). The preneoplastic setting of the cirrhotic background provides a conducive environment for cellular transformation. The role of classical inflammation in cirrhosis is widely known, but the exact mechanism linking inflammation and cancer remains elusive. Recent studies have elucidated roles for NF-κB, STAT3 and JNK as possible missing links. In addition, the "inflammasome" (a multiprotein complex and sensor of cellular damage) is a recently identified player in this field. The hallmarks of cirrhosis include necroinflammation, deposition of extracellular matrix and shortening of telomeres, leading to senescence and regeneration. Additionally, the accumulation of genetic/epigenetic changes propels atypical cells toward a malignant phenotype. This review provides recent information on the classical inflammatory pathway, together with a spotlight on inflammasomes and the immunomodulatory role of cellular senescence during the progression from cirrhosis to HCC. Moreover, lacunae in the current knowledge were identified and key questions raised on whether the observed adaptive responses are beneficial or detrimental to tissue homeostasis in a complex organ like liver.

Analysis of Notch and TGF-ß Signaling Expression in Different Stages of Disease Progression During Hepatitis B Virus Infection.

OBJECTIVES: CD4+ regulatory T cells (Tregs) seem to have a key role in persistence of hepatitis B virus (HBV) infection. Notch and transforming growth factor (TGF-ß) signaling independently help in the differentiation and regulation of CD4+T cells, including T-helper (T(H)) 1, T(H)2, and Tregs. Whether, the two pathways have modulatory role on different stages of HBV infection and severity of liver disease is not clear. We investigated Notch and TGF-ß families' gene expression in peripheral blood and intrahepatic lymphocytes in patients with different stages of chronic HBV (CHB) infection. METHODS: Peripheral blood mononuclear cells (PBMCs), CD4(+), and CD8(+) T cells were isolated from patients with acute HBV (AVH-B, n=15), CHB (n=16), and controls (HC, n=10). In addition to PBMCs, intrahepatic lymphocytes were obtained from liver biopsies from CHB (n=12), cirrhosis (n=12), hepatocellular carcinoma (HCC, n=5), and healthy livers (n=5). Notch family (Notch1-4, Hes1, Jag1, and NF-kß) and TGF-ß family gene expressions were studied by real-time PCR, flow cytometry, and immunohistochemistry. RESULTS: Relative expression of Notch signaling target genes, Hes1 and NF-kß, was higher in the total PBMCs of AVH-B and CHB patients than that in HC patients (Log relative quantification (RQ); 1.1 AVH-B vs. 0.3 HC, 1.3 CHB vs. 0.3 HC; P=0.02). CD8(+) T cells showed upregulated expression of Hes1 and Notch1 (P=0.02 and 0.01, respectively) in AVH-B than in CHB patients. Also, in AVH-B patients, HBV-specific CD8(+) T-cell proliferation (5.74% vs. 2.7%) and TGF-ß signaling activity were higher. All Notch receptors and ligands were upregulated in the PBMCs in CHB infection (CHB vs. cirrhosis, P=0.001; CHB vs. HCC, P=0.023; and cirrhosis vs. HCC, P=NS). Intrahepatic expression of Notch1 and FoxP3 were significantly higher in cirrhotics and HCCs, and further blockage of Notch signaling reduced the FoxP3 expression. Array data of TGF-ß family showed increased TGF-ß3, TGF-α, SMAD3, SMAD4, SMAD6, and GDF9 expression on intrahepatic lymphocytes in cirrhotic and HCC patients compared with CHB. CONCLUSIONS: Our findings suggest that there is a complementary association between Notch1 and Hes1 in CD8(+)T cells during AVH-B infection. On development of CHB infection, repression of the Notch receptors mediates the regulation of immune response in patients, who progress to cirrhosis and HCC. Finally, HBV infection drives increased Notch1, TGF-ß, and FoxP3 expression on intrahepatic T cells in cirrhosis, resulting in fibrogenesis and disease progression.