NRDE2 deficiency impairs homologous recombination repair and sensitizes hepatocellular carcinoma to PARP inhibitors.

To identify novel susceptibility genes for hepatocellular carcinoma (HCC), we performed a rare-variant association study in Chinese populations consisting of 2,750 cases and 4,153 controls. We identified four HCC-associated genes, including NRDE2, RANBP17, RTEL1, and STEAP3. Using NRDE2 (index rs199890497 [p.N377I], p = 1.19 × 10-9) as an exemplary candidate, we demonstrated that it promotes homologous recombination (HR) repair and suppresses HCC. Mechanistically, NRDE2 binds to the subunits of casein kinase 2 (CK2) and facilitates the assembly and activity of the CK2 holoenzyme. This NRDE2-mediated enhancement of CK2 activity increases the phosphorylation of MDC1 and then facilitates the HR repair. These functions are eliminated almost completely by the NRDE2-p.N377I variant, which sensitizes the HCC cells to poly(ADP-ribose) polymerase (PARP) inhibitors, especially when combined with chemotherapy. Collectively, our findings highlight the relevance of the rare variants to genetic susceptibility to HCC, which would be helpful for the precise treatment of this malignancy.

Establishment of organoid models for pancreatic ductal adenocarcinoma and screening of individualized therapy strategy.

BACKGROUND: Patients with pancreatic ductal adenocarcinoma (PDAC) who undergo surgical resection and receive effective chemotherapy have the best chance for long-term survival. Unfortunately, because of the heterogeneity of pancreatic cancer, it is difficult to find a personalized treatment strategy for patients. Organoids are ideal preclinical models for personalized medicine. Therefore, we explore the cultivation conditions and construction methods of PDAC organoid models to screen the individualized therapy strategy. METHODS: Fresh PDAC tissues from surgical resection were collected and digested with digestive enzymes; then the tumor cells were embedded in Matrigel with a suitable medium to establish the PDAC organoid models. The genetic stability of the organoids was analyzed using whole exon sequencing; hematoxylin and eosin staining and immunohistochemistry of organoids were performed to analyze their consistency with the pathological morphology of the patient's tumor tissue; After 2 days of organoid culture, we selected four commonly used clinical chemotherapy drugs for single or combined treatment to analyze drug sensitivity. RESULTS: Two cases of PDAC organoid models were successfully established, and the results of their pathological characteristics and exome sequencing were consistent with those of the patient's tumor tissue. Both PDAC organoids showed more sensitivity to gemcitabine and cisplatin, and the combined treatment was more effective than monotherapy. CONCLUSION: Both organoids better retained the pathological characteristics, genomic stability, and heterogeneity with the original tumor. Individual PDAC organoids exhibited different sensitivities to the same drugs. Thus, this study provided ideal experimental models for screening individualized therapy strategy for patients with PDAC.

Near-infrared fluorescence imaging of hepatocellular carcinoma cells regulated by ß-catenin signaling pathway.

Background: Near-infrared fluorescence (NIRF) imaging has recently emerged as a promising tool for noninvasive cancer imaging. However, lack of tumor sensitivity and specificity restricts the application of NIRF dyes in surgical navigation. Methods: Herein, we investigated the imaging features of NIRF dye MHI-148 and indocyanine green (ICG) in live cell imaging and xenograft nude mice models. TCGA dataset analysis and immunohistochemistry were conducted to investigate the expression of OATPs or ABCGs in hepatocellular carcinoma (HCC) tissues. OATPs or ABCGs were knocked down and overexpressed in HCC cells using transient transfection by siRNA and plasmids or stable transfection by lentivirus. Further, qRT-PCR ,Western blotting and the use of agonists or inhibitors targeting ß-catenin signaling pathway were applied to explore its important role in regulation of OATP2B1 and ABCG2 expression. Results: Here we demonstrated that NIRF dye MHI-148 was biocompatible as indocyanine green (ICG) but with higher imaging intensity and preferential uptake and retention in hepatocellular carcinoma (HCC) cells and tissues. Moreover, our data indicated that membrane transporters OATP2B1 and ABCG2, which regulated by ß-catenin signaling pathway, mediated tumor-specific accumulation and retention of MHI-148 in HCC cells. In addition, the treatment with ß-catenin inhibitor significantly enhanced the accumulation of MHI-148 in HCC tissues and improved the efficacy of tumor imaging with MHI-148 in vivo. Conclusions: Our study uncovers a mechanism that links the distribution and expression of the membrane transporters OATP2B1 and ABCG2 to the tumor-specific accumulation of MHI-148, and provides evidence supporting a regulating role of the ß-catenin signaling pathway in OATP2B1 and ABCG2- induced retention of MHI-148 inHCC tissues, and strategy targeting key components of MHI-148 transport machinery may be a potential approach to improve HCC imaging.

Down-regulation of BMAL1 by MiR-494-3p Promotes Hepatocellular Carcinoma Growth and Metastasis by Increasing GPAM-mediated Lipid Biosynthesis.

The circadian clock confers daily rhythmicity to many crucial biological processes and behaviors and its disruption is closely associated with carcinogenesis in several types of cancer. Brain and muscle arnt-like protein 1 (BMAL1) is a core circadian rhythm component in mammals and its dysregulation has been shown to contribute to aberrant metabolism in human diseases. However, the biological functions of BMAL1, especially its involvement in aberrant lipid metabolism in hepatocellular carcinoma (HCC), remain elusive. In the present study, we found that BMAL1 was frequently down-regulated in HCC cells mainly due to the up-regulation of miR-494-3p. Down-regulation of BMAL1 was significantly associated with poor survival in HCC patients. BMAL1 down-regulation promoted HCC cell growth and metastasis both in vitro and in vivo. Mechanistically, through cooperating with EZH2, BMAL1 transcriptionally suppressed the expression of glycerol-3-phosphate acyltransferase mitochondrial (GPAM), a key enzyme involved in the regulation of lipid biosynthesis, leading to reduced levels lysophosphatidic acid (LPA), which have long been known as mediator of oncogenesis. Particularly, treatment with SR8278, an activator of BMAL1, exhibited a therapeutic effect on HCC in vitro and in vivo. In conclusion, BMAL1 plays a critical anti-oncogenic role in HCC, providing strong research evidence for BMAL1 as a prospective target for HCC therapy.

The Application of [68Ga]-Labeled FAPI-04 PET/CT for Targeting and Early Detection of Pancreatic Carcinoma in Patient-Derived Orthotopic Xenograft Models.

[18F]FDG as a probe of PET/CT is a radiolabeled glucose analogue taken up by most cells, but its batch activity is limited. [68Ga]FAPI-04 is a promising alternative based on a fibroblast activation protein-specific inhibitor (FAPI) labeled with radiotracer FAP. Here, a series of databases suggested that FAP expression was significantly different in pancreatic cancer compared to normal tissue. The FAP-positive fibroblasts were evaluated around the tumor cells and the stroma. A patient-derived orthotopic xenograft (PDOX) model of pancreatic adenocarcinoma (PDAC) exhibits significantly higher quantitative uptake of [68Ga]FAPI-04 (P < 0.05) than [18F]FDG PET/CT in various organs. Because of relatively high (T/M) ratios, the [68Ga]FAPI-04 is excellent for B-mode ultrasound, NIRF, and PET/CT. Thus, [68Ga]FAPI-04 PET displayed a better tumor specificity and can be a potential application for the early detection of pancreatic cancer.

A computational framework to unify orthogonal information in DNA methylation and copy number aberrations in cell-free DNA for early cancer detection.

Cell-free DNA (cfDNA) provides a convenient diagnosis avenue for noninvasive cancer detection. The current methods are focused on identifying circulating tumor DNA (ctDNA)s genomic aberrations, e.g. mutations, copy number aberrations (CNAs) or methylation changes. In this study, we report a new computational method that unifies two orthogonal pieces of information, namely methylation and CNAs, derived from whole-genome bisulfite sequencing (WGBS) data to quantify low tumor content in cfDNA. It implements a Bayes model to enrich ctDNA from WGBS data based on hypomethylation haplotypes, and subsequently, models CNAs for cancer detection. We generated WGBS data in a total of 262 samples, including high-depth (>20×, deduped high mapping quality reads) data in 76 samples with matched triplets (tumor, adjacent normal and cfDNA) and low-depth (~2.5×, deduped high mapping quality reads) data in 186 samples. We identified a total of 54 Mb regions of hypomethylation haplotypes for model building, a vast majority of which are not covered in the HumanMethylation450 arrays. We showed that our model is able to substantially enrich ctDNA reads (tens of folds), with clearly elevated CNAs that faithfully match the CNAs in the paired tumor samples. In the 19 hepatocellular carcinoma cfDNA samples, the estimated enrichment is as high as 16 fold, and in the simulation data, it can achieve over 30-fold enrichment for a ctDNA level of 0.5% with a sequencing depth of 600×. We also found that these hypomethylation regions are also shared among many cancer types, thus demonstrating the potential of our framework for pancancer early detection.

Lipocalin 2 may be a key factor regulating the chemosensitivity of pancreatic cancer to gemcitabine.

Owing to the high heterogeneity of pancreatic cancer, patient-derived xenografts (PDX) can compensate for the defects of cell line-derived xenografts (CDX) and also better preserve the heterogeneity and tumor microenvironment of primary tumors. Further, gemcitabine, which is used for the treatment of various cancers, is prone to tumor drug resistance, and this limits its sustained efficacy. Therefore, in this study, our objective was to screen appropriate individual therapeutic drugs for pancreatic cancer. To this end, we established pancreatic cancer PDX models from different patients and screened gemcitabine sensitivity regulatory molecules via high-throughput transcriptome sequencing and bioinformatics analysis. Based on the results obtained, gemcitabine was identified as the most suitable chemotherapeutic drug in a variety of PDX models. Additionally, our results indicated that Lipocalin 2 (LCN 2) may play an important role in the sensitivity of pancreatic cancer to gemcitabine treatment. Thus, the study provides a new potential intervention target for the treatment of pancreatic cancer in clinical practice.

Mitochondrial fragmentation is crucial for c-Myc-driven hepatoblastoma-like liver tumors.

Hepatoblastoma is the most common liver cancer in children, and the aggressive subtype often has a poor prognosis and lacks effective targeted therapy. Although aggressive hepatoblastoma (HB) is often accompanied by abnormally high expression of the transcription factor c-Myc, the underlying mechanism remains unclear. In this study, we found that mitochondrial fragmentation was enhanced by c-Myc overexpression in human aggressive HB tissues and was associated with poor prognosis. Then, a mouse model resembling human HB was established via hydrodynamic injection of c-Myc plasmids. We observed that liver-specific knockout of the mitochondrial fusion molecule MFN1 or overexpression of mitochondrial fission molecule DRP1 promoted the occurrence of c-Myc-driven liver cancer. In contrast, when MFN1 was overexpressed in the liver, tumor formation was delayed. In vitro experiments showed that c-Myc transcriptionally upregulated the expression of DRP1 and decreased MFN1 expression through upregulation of miR-373-3p. Moreover, enhanced mitochondrial fragmentation significantly promoted aerobic glycolysis and the proliferation of HB cells by significantly increasing reactive oxygen species (ROS) production and activating the RAC-alpha serine/threonine-protein kinase (AKT)/mammalian target of rapamycin (mTOR) and nuclear factor κB (NF-κB) pathways. Taken together, our results indicate that c-Myc-mediated mitochondrial fragmentation promotes the malignant transformation and progression of HB by activating ROS-mediated multi-oncogenic signaling.

Screening of an individualized treatment strategy for an advanced gallbladder cancer using patient-derived tumor xenograft and organoid models.

Gallbladder cancer is a highly aggressive malignancy with poor sensitivity to postoperative radiotherapy or chemotherapy; therefore, the development of individualized treatment strategies is paramount to improve patient outcomes. Both patient-derived tumor xenograft (PDX) and patient-derived tumor organoid (PDO) models derived from surgical specimens can better preserve the biological characteristics and heterogeneity of individual original tumors, display a unique advantage for individualized therapy and predicting clinical outcomes. In this study, PDX and PDO models of advanced gallbladder cancer were established, and the consistency of biological characteristics between them and primary patient samples was confirmed using pathological analysis and RNA-sequencing. Additionally, we tested the efficacy of chemotherapeutic drugs, targeted drugs, and immune checkpoint inhibitors using these two models. The results demonstrated that gemcitabine combined with cisplatin induced significant therapeutic effects. Furthermore, treatment with immune checkpoint inhibitors elicited promising responses in both the humanized mice and PDO immune models. Based on these results, gemcitabine combined with cisplatin was used for basic treatment, and immune checkpoint inhibitors were applied as a complementary intervention for gallbladder cancer. The patient responded well to treatment and exhibited a clearance of tumor foci. Our findings indicate that the combined use of PDO and PDX models can guide the clinical treatment course for gallbladder cancer patients to achieve individualized and effective treatment.

Low Expression of Phosphodiesterase 2 (PDE2A) Promotes the Progression by Regulating Mitochondrial Morphology and ATP Content and Predicts Poor Prognosis in Hepatocellular Carcinoma.

Phosphodiesterase 2 (PDE2A) modulates the levels of cAMP/cGMP and was recently found to be involved in mitochondria function regulation, closely related to multiple types of tumor progression. This study aimed to estimate the prognostic significance and biological effects of PDE2A on hepatocellular carcinoma (HCC). We comprehensively analyzed the PDE2A mRNA expression in HCC based on The Cancer Genome Atlas (TCGA) database and investigated the effects of PDE2A on the proliferation and metastatic capacity of HCC cells. PDE2A was downregulated in 25 cancer types, including HCC. Lower PDE2A expression was a protective factor in HCC and was negatively associated with serum AFP levels, tumor status, vascular invasion, histologic grade, and pathologic stage of HCC. Moreover, tumors with low PDE2A expression displayed a decreased immune function. Then, the ROC curve was used to assess the diagnostic ability of PDE2A in HCC (AUC = 0.823 in TCGA and AUC = 0.901 in GSE76427). Patients with low PDE2A expression exhibited worse outcomes compared with those with high PDE2A expression. Additionally, GO functional annotations demonstrated the involvement of PDE2A in the ECM organization, systems development, and ERK-related pathways, indicating that PDE2A might regulate HCC growth and metastasis. The in vitro experiments confirmed that overexpression of PDE2A inhibited proliferation, colony formation, migration, and invasion in two HCC cell lines (HLF and SNU-368), while inhibition of PDE2A has the opposite results. The mechanism of PDE2A's effect on HCC cells is attributed to the change of mitochondrial morphology and ATP content. These data demonstrated that PDE2A closely participated in the regulation of HCC proliferation and metastasis and can be used as a predictive marker candidate and a potential therapeutic target for HCC.

Genetic variants in NPAS2 gene and clinical outcomes of resectable non-small-cell lung cancer.

Background: A series of studies have demonstrated that NPAS2 plays a critical role in the development and progression of several cancers. However, the association between genetic variants in the NPAS2 gene and the clinical outcome of patients with non-small-cell lung cancer (NSCLC) has not been investigated. Methods: Six functional SNPs in NPAS2 were selected and genotyped using the Sequenom iPLEX genotyping system in a cohort of 484 Chinese NSCLC patients undergoing surgery. Multivariate Cox proportional hazards model were used for the prognosis analysis. Results: We found that SNP rs2305158 exhibited a significant association with overall survival of NSCLC patients in the dominant model (hazard ratio [HR]: 0.68; 95% CI: 0.49-0.95; p = 0.02). Lymph node metastasis was significantly associated with increased death risk (HR: 1.73; 95% CI: 1.24-2.40; p = 0.001) in patients with the homozygous wildtype (WW) genotype of rs2305158. However, no significant association was observed between them in patients carrying a heterozygous variant (WV) or homozygous variant (VV) genotype of rs2305158. Finally, in the joint and interaction analysis, the patients carrying homozygous wildtype (WW) genotype and lymph node metastasis from N1 to N3 conferred a significant increased effect on death (HR: 2.29; 95% CI: 1.40-3.76; p = 0.001). Conclusions: Our results suggest that NPAS2 polymorphisms may serve as an independent prognostic marker for NSCLC patients.

Next generation sequencing-based analysis of mitochondrial DNA characteristics in plasma extracellular vesicles of patients with hepatocellular carcinoma.

Emerging evidence has revealed that mitochondrial DNA (mtDNA) is encapsulated in plasma extracellular vesicles (EVs). However, the characteristics of mtDNA in EVs from patients with cancer remain largely unexplored, which greatly limits its clinical application. Whole genome and capture-based sequencing found that EV mtDNA covered the whole mitochondrial genome. The medium fragment size in EV mtDNA was significantly larger compared with that in cell-free mtDNA [cfmtDNA; 159 vs. 109 base pairs (bp); P<0.001]. EV DNA appeared to have a higher mtDNA copy number compared with cfDNA. Of note, patients with hepatitis had >300-bp fragments in EV mtDNA compared with patients with hepatocellular carcinoma (HCC) and healthy controls. EV mtDNA fragments >300 bp in length exhibited a significantly higher proportion of EV mtDNA fragment ends than those that were ≤300 bp in length in patients with hepatitis. The EV mtDNA copy number in patients with HCC and hepatitis were significantly lower compared with those in healthy controls. Furthermore, inconsistencies in the mtDNA heteroplasmic variant were observed among HCC tissues, plasma and EVs. In conclusion, EV mtDNA exhibited different characteristics among patients with HCC, hepatitis and healthy controls, indicating the potential value of EV mtDNA as a diagnostic biomarker that complements cfmtDNA.

Noncoding telomeric repeat-containing RNA inhibits the progression of hepatocellular carcinoma by regulating telomerase-mediated telomere length.

Telomeric repeat-containing RNA (TERRA) is closely involved in the regulation of telomere length, which plays critical roles in tumorigenesis. However, the biological significance of TERRA in hepatocellular carcinoma (HCC) remains largely unknown. In this study, we found that HCC cells show a frequent downregulation of TERRA and its positive regulator TTAGGG repeat binding factor-1 (TRF1), whereas the negative regulator TTAGGG repeat binding factor-1 (TRF2) was upregulated. We found that TERRA, TRF1, and TRF2 contributed to poor prognosis of HCC patients. Importantly, we found that the downregulation of TERRA significantly promoted HCC cell growth and metastasis in vitro and in vivo, whereas the upregulation of TERRA showed an opposite effect. Mechanistically, downregulation of TERRA significantly increased telomerase activity and promoted telomere elongation. Moreover, the inhibitory effects of TERRA overexpression on the growth and metastasis of HCC cells were reversed by treatment with TA-65 that activates telomerase activity. In contrast, the protumor effect of TERRA downregulation was reversed by treatment with TMPyP4 that inhibits telomerase activity. Our findings reveal that TERRA plays a critical role in HCC cell growth and metastasis, indicating that TERRA is a potential therapeutic target for HCC.

Mediated Imaging and Improved Targeting of Farnesylthiosalicylic Acid Delivery for Pancreatic Cancer via Conjugation with Near-Infrared Fluorescence Heptamethine Carbocyanine Dye.

S-trans-trans-Farnesylthiosalicylic acid (FTS) is a Ras inhibitor that exhibits desirable anticancer property and currently undergoing clinical trials for pancreatic cancer (PC). However, its poor water solubility and low bioavailability have severely hampered clinical applications. A strategy to improve FTS bioavailability is to develop a suitable drug delivery method. Here, we use a near-infrared fluorescence (NIRF) heptamethine carbocyanine (HC) dye conjugated with FTS (to produce FTS-148) as a drug delivery system to enhance FTS bioavailability. We further investigate its tumor-targeting functions. FTS-148 displayed better bioavailability and photophysical property and selective recognition of cancer cells. FTS-148 significantly reduced PC cell proliferation, and more effective than FTS in restricting tumor growth both in a cell-derived xenograft (CDX) model and a patient-derived tumor xenograft (PDX) model. FTS-148 can specifically recognize PC cells in mice subcutaneous models or rabbit orthotopic models and allows real-time monitoring of the therapeutic effects by NIRF optical imaging. FTS-148 treatment significantly reduced Ras expression in PC cells and increased tumor tissue apoptosis. In short, FTS conjugated with HC dye had enhanced bioavailability and tumor-targeting property. It provides a potential agent for imaging and therapy of PC.

Upregulation of histamine receptor H1 promotes tumor progression and contributes to poor prognosis in hepatocellular carcinoma.

H1 histamine receptor (H1HR) belongs to the family of rhodopsin-like G-protein-coupled receptors. Recent studies have shown that H1HR expression is increased in several types of cancer. However, its functional roles in tumor progression remain largely unknown, especially in hepatocellular carcinoma (HCC). We found that H1HR is frequently unregulated in HCC, which is significantly associated with both recurrence-free survival and overall survival in HCC patients. Functional experiments revealed that H1HR promoted both the growth and metastasis of HCC cells by inducing cell cycle progression, formation of lamellipodia, production of matrix metalloproteinase 2, and suppression of cell apoptosis. Activation of cyclic adenosine monophosphate-dependent protein kinase A was found to be involved in H1HR-mediated HCC cell growth and metastasis. In addition, we found that overexpression of H1HR was mainly due to the downregulation of miR-940 in HCC cells. Moreover, the H1HR inhibitor terfenadine significantly suppressed tumor growth and metastasis in an HCC xenograft nude mice model. Our findings demonstrate that H1HR plays a critical role in the growth and metastasis of HCC cells, which provides experimental evidence supporting H1HR as a potential drug target for the treatment of HCC.

NIRF Optical/PET Dual-Modal Imaging of Hepatocellular Carcinoma Using Heptamethine Carbocyanine Dye.

Combining near-infrared fluorescence (NIRF) and nuclear imaging techniques provides a novel approach for hepatocellular carcinoma (HCC) diagnosis. Here, we report the synthesis and characteristics of a dual-modality NIRF optical/positron emission tomography (PET) imaging probe using heptamethine carbocyanine dye and verify its feasibility in both nude mice and rabbits with orthotopic xenograft liver cancer. This dye, MHI-148, is an effective cancer-specific NIRF imaging agent and shows preferential uptake and retention in liver cancer. The corresponding NIRF imaging intensity reaches 109/cm2 tumor area at 24 h after injection in mice with HCC subcutaneous tumors. The dye can be further conjugated with radionuclide 68Ga (68Ga-MHI-148) for PET tracing. We applied the dual-modality methodology toward the detection of HCC in both patient-derived orthotopic xenograft (PDX) models and rabbit orthotopic transplantation models. NIRF/PET images showed clear tumor delineation after probe injection (MHI-148 and 68Ga-MHI-148). The tumor-to-muscle (T/M) standardized uptake value (SUV) ratios were obtained from PET at 1 h after injection of 68Ga-MHI-148, which was helpful for effectively capturing small tumors in mice (0.5 cm × 0.3 cm) and rabbits (1.2 cm × 1.8 cm). This cancer-targeting NIRF/PET dual-modality imaging probe provides a proof of principle for noninvasive detection of deep-tissue tumors in mouse and rabbit and is a promising technique for more accurate and early detection of HCC.

Genome-Wide Association Study Identifies a New Locus at 7q21.13 Associated with Hepatitis B Virus-Related Hepatocellular Carcinoma.

Purpose: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. In China, chronic hepatitis B virus (HBV) infection remains the major risk factor for HCC. In this study, we performed a genome-wide association study (GWAS) among Chinese populations to identify novel genetic loci contributing to susceptibility to HBV-related HCC.Experimental Design: GWAS scan is performed in a collection of 205 HBV-related HCC trios (each trio includes an affected proband and his/her both parents), and 355 chronic HBV carriers with HCC (cases) and 360 chronic HBV carriers without HCC (controls), followed by two rounds of replication studies totally consisting of 3,796 cases and 2,544 controls.Results: We identified a novel association signal within the CDK14 gene at 7q21.13 (index rs10272859, OR = 1.28, P = 9.46 × 10-10). Furthermore, we observed that the at-risk rs10272859[G] allele was significantly associated with higher mRNA expression levels of CDK14 in liver tissues. Chromosome conformation capture assays in liver cells confirmed that a physical interaction exists between the promoter region of CDK14 and the risk-associated SNPs in strong linkage disequilibrium with the index rs10272859 at 7q21.13. This index rs10272859 also showed significant association with the survival of HCC patients.Conclusions: Our findings highlight a novel locus at 7q21.13 conferring both susceptibility and prognosis to HBV-related HCC, and suggest the CDK14 gene to be the functional target of the 7q21.13 locus. Clin Cancer Res; 24(4); 906-15. ©2017 AACR.

Heptamethine carbocyanine DZ-1 dye for near-infrared fluorescence imaging of hepatocellular carcinoma.

Near-infrared fluorescence (NIRF) dyes have recently emerged as promising tools for non-invasive imaging of different types of cancers. Here, we explored the potential utility of a NIRF DZ-1 dye, with dual imaging and tumour targeting functions, in hepatocellular carcinoma (HCC). We showed the preferential uptake of DZ-1 by HCC cells in vitro and in derived subcutaneous/orthotopic tumour xenografts, accompanied by a minimal effect on normal cells. DZ-1 simplified tumour growth profiling as well, since we were able to correlate NIRF signals with tumour volume and/or tumour-emitting luminescence in mice. Using both orthotopic tumour transplantation and cirrhosis models in parallel, we demonstrated the ability of DZ-1 to differentiate liver tumour from cirrhosis. DZ-1 showed superiority in HCC imaging over indocyanine green by demonstrating significantly enhanced tumour-targeting specificity. At the cellular level, DZ-1 was mainly retained in mitochondria and lysosomes. Additionally, DZ-1 fluorescence spectroscopy has been used for the intraoperative navigation of rabbit liver cancer, to determine surgical margins. We showed that tumor hypoxia and select organic anion-transporting polypeptide genes mediate NIRF dye uptake in HCC, which was supported by clinical evidence. All these findings represent the first evidence that DZ-1 is an effective molecular probe for tumour-specific imaging in HCC, and provide insights into the development of a new generation of imaging agents for intraoperative guidance of cancer surgery.

Mitochondrial fission forms a positive feedback loop with cytosolic calcium signaling pathway to promote autophagy in hepatocellular carcinoma cells.

Both mitochondrial morphology and the level of cytosolic calcium [Ca2+]c are actively changed and play critical roles in a number of malignancies. However, whether communications existed between these two processes to ingeniously control the malignant phenotype are far from clear. We investigated the reciprocal regulation between mitochondrial fission and cytosolic calcium signaling in human hepatocellular carcinoma (HCC) cells. Furthermore, the underlying molecular mechanisms and the synergistic effect on autophagy were explored. Our results showed that mitochondrial fission increased the [Ca2+]c and calcium oscillation in HCC cells. We further found that mitochondrial fission-mediated calcium signaling was dependent on ROS-activated NF-κB pathways, which facilitated the expression of STIM1 and subsequent store-operated calciumentry. Additionally, we also demonstrated that increase in [Ca2+]c promoted mitochondrial fission by up-regulating expression of Drp1 and FIS1 via transcription factors NFATC2 and c-Myc, respectively. Moreover, the positive feedback loop significantly promoted HCC cell global autophagy by Ca2+/CAMKK/AMPK pathway. Our data demonstrate a positive feedback loop between mitochondrial fission and cytosolic calcium signaling and their promoting role in autophagy of HCC cells, which provides evidence for this loop as a potential drug target in tumor treatment.

Genetic variations of mitochondrial genome modify risk and prognosis of hepatocellular carcinoma patients.

BACKGROUND: Previous studies have indicated that mitochondrial genetic variations were associated with the risk of many cancers. However, there are few reports on the association between single nucleotide polymorphisms (SNPs) or haplogroups of mitochondrial DNA (mtDNA) and the risk or prognosis of hepatocellular carcinoma (HCC). METHODS: In order to investigate the predictive and prognostic role of mtDNA SNPs and haplogroups in HCC, the mitochondrial genome of 188 HCC patients and 344 healthy controls were sequenced by next generation sequencing technology. Then, logistic regression analysis was used to determine the effect of mtDNA SNP or haplogroup on risk and prognosis of HCC patients. RESULTS: The haplogroup M7 had an odds ratio (OR) of 0.47 (95% CI=0.24-0.91; P=0.026) to develop HCC. The frequency of 152T/C, 199T/C, 4048G/A, 9824T/C, 15784T/C, 16185C/T and 16399A/G was significantly different between HCC patients and the controls. In addition, multivariate analysis with COX hazards model showed that the patients with haplogroup M8 had lower survival rate than the patients with haplogroup D4 (HR=2.62, 95% CI=1.03-6.68; P=0.044). Three SNPs 15784T/C, 16185C/T and 16399A/G were also identified to have a statistically significant association with postoperative survival in HCC. CONCLUSIONS: To date, these results provide the first evidence that mtDNA SNPs and haplogroups may be potential risk factors for susceptibility and survival of HCC patients.