Fibroblast Activation Protein α-Directed Imaging and Therapy of Solitary Fibrous Tumor.

Fibroblast activation protein α (FAPα) is expressed at high levels in several types of tumors. Here, we report the expression pattern of FAPα in solitary fibrous tumor (SFT) and its potential use as a radiotheranostic target. Methods: We analyzed FAPα messenger RNA and protein expression in biopsy samples from SFT patients using immunohistochemistry and multiplexed immunofluorescence. Tracer uptake and detection efficacy were assessed in patients undergoing clinical 68Ga-FAPα inhibitor (FAPI)-46 PET,18F-FDG PET, and contrast-enhanced CT. 90Y-FAPI-46 radioligand therapy was offered to eligible patients with progressive SFT. Results: Among 813 patients and 126 tumor entities analyzed from the prospective observational MASTER program of the German Cancer Consortium, SFT (n = 34) had the highest median FAPα messenger RNA expression. Protein expression was confirmed in tumor biopsies from 29 of 38 SFT patients (76%) in an independent cohort. Most cases showed intermediate to high FAPα expression by immunohistochemistry (24/38 samples, 63%), which was located primarily on the tumor cell surface. Nineteen patients who underwent 68Ga-FAPI-46 PET imaging demonstrated significantly increased tumor uptake, with an SUVmax of 13.2 (interquartile range [IQR], 10.2), and an improved mean detection efficacy of 94.5% (SEM, 4.2%), as compared with 18F-FDG PET (SUVmax, 3.2 [IQR, 3.1]; detection efficacy, 77.3% [SEM, 5.5%]). Eleven patients received a total of 34 cycles (median, 3 cycles [IQR, 2 cycles]) of 90Y-FAPI-46 radioligand therapy, which resulted in disease control in 9 patients (82%). Median progression-free survival was 227 d (IQR, 220 d). Conclusion: FAPα is highly expressed by SFT and may serve as a target for imaging and therapy. Further studies are warranted to define the role of FAPα-directed theranostics in the care of SFT patients.

Coinhibition of topoisomerase 1 and BRD4-mediated pause release selectively kills pancreatic cancer via readthrough transcription.

Pancreatic carcinoma lacks effective therapeutic strategies resulting in poor prognosis. Transcriptional dysregulation due to alterations in KRAS and MYC affects initiation, development, and survival of this tumor type. Using patient-derived xenografts of KRAS- and MYC-driven pancreatic carcinoma, we show that coinhibition of topoisomerase 1 (TOP1) and bromodomain-containing protein 4 (BRD4) synergistically induces tumor regression by targeting promoter pause release. Comparing the nascent transcriptome with the recruitment of elongation and termination factors, we found that coinhibition of TOP1 and BRD4 disrupts recruitment of transcription termination factors. Thus, RNA polymerases transcribe downstream of genes for hundreds of kilobases leading to readthrough transcription. This occurs during replication, perturbing replisome progression and inducing DNA damage. The synergistic effect of TOP1 + BRD4 inhibition is specific to cancer cells leaving normal cells unaffected, highlighting the tumor's vulnerability to transcriptional defects. This preclinical study provides a mechanistic understanding of the benefit of combining TOP1 and BRD4 inhibitors to treat pancreatic carcinomas addicted to oncogenic drivers of transcription and replication.

Fibroblast Activation Protein-Targeting Minibody-IRDye700DX for Ablation of the Cancer-Associated Fibroblast with Photodynamic Therapy.

Fibroblast activation protein (FAP), expressed on cancer-associated fibroblasts, is a target for diagnosis and therapy in multiple tumour types. Strategies to systemically deplete FAP-expressing cells show efficacy; however, these induce toxicities, as FAP-expressing cells are found in normal tissues. FAP-targeted photodynamic therapy offers a solution, as it acts only locally and upon activation. Here, a FAP-binding minibody was conjugated to the chelator diethylenetriaminepentaacetic acid (DTPA) and the photosensitizer IRDye700DX (DTPA-700DX-MB). DTPA-700DX-MB showed efficient binding to FAP-overexpressing 3T3 murine fibroblasts (3T3-FAP) and induced the protein's dose-dependent cytotoxicity upon light exposure. Biodistribution of DTPA-700DX-MB in mice carrying either subcutaneous or orthotopic tumours of murine pancreatic ductal adenocarcinoma cells (PDAC299) showed maximal tumour uptake of 111In-labelled DTPA-700DX-MB at 24 h post injection. Co-injection with an excess DTPA-700DX-MB reduced uptake, and autoradiography correlated with FAP expression in the stromal tumour region. Finally, in vivo therapeutic efficacy was determined in two simultaneous subcutaneous PDAC299 tumours; only one was treated with 690 nm light. Upregulation of an apoptosis marker was only observed in the treated tumours. In conclusion, DTPA-700DX-MB binds to FAP-expressing cells and targets PDAC299 tumours in mice with good signal-to-background ratios. Furthermore, the induced apoptosis indicates the feasibility of targeted depletion of FAP-expressing cells with photodynamic therapy.

Fibroblast Activation Protein-Targeted Photodynamic Therapy of Cancer-Associated Fibroblasts in Murine Models for Pancreatic Ductal Adenocarcinoma.

Patients with pancreatic ductal adenocarcinoma (PDAC) have a dismal 5 year survival of 9%. One important limiting factor for treatment efficacy is the dense tumor-supporting stroma. The cancer-associated fibroblasts in this stroma deposit excessive amounts of extracellular matrix components and anti-inflammatory mediators, which hampers the efficacy of chemo- and immunotherapies. Systemic depletion of all activated fibroblasts is, however, not feasible nor desirable and therefore a local approach should be pursued. Here, we provide a proof-of-principle of using fibroblast activation protein (FAP)-targeted photodynamic therapy (tPDT) to treat PDAC. FAP-targeting antibody 28H1 and irrelevant control antibody DP47GS were conjugated to the photosensitizer IRDye700DX (700DX) and the chelator diethylenetriaminepentaacetic acid. In vitro binding and cytotoxicity were evaluated using the fibroblast cell-line NIH-3T3 stably transfected with FAP. Biodistribution of 111In-labeled antibody-700DX constructs was determined in mice carrying syngeneic tumors of the murine PDAC cell line PDAC299, and in a genetically engineered PDAC mouse model (CKP). Then, tPDT was performed by exposing the subcutaneous or the spontaneous PDAC tumors to 690 nm light. Induction of apoptosis after treatment was assessed using automated analyses of immunohistochemistry for cleaved caspase-3. 28H1-700DX effectively bound to 3T3-FAP cells and induced cytotoxicity upon exposure to 690 nm light, whereas no binding or cytotoxic effects were observed for DP47GS-700DX. Although both 28H1-700DX and DP47GS-700DX accumulated in subcutaneous PDAC299 tumors, autoradiography demonstrated that only 28H1-700DX reached the tumor core. On the contrary, control antibody DP47GS-700DX was only present at the tumor rim. In CKP mice, both antibodies accumulated in the tumor, but tumor-to-blood ratios of 28H1-700DX were higher than that of the control. Notably, in vivo FAP-tPDT caused upregulation of cleaved caspase-3 staining in both subcutaneous and in spontaneous tumors. In conclusion, we have shown that tPDT is a feasible approach for local depletion of FAP-expressing stromal cells in murine models for PDAC.

Superior Tumor Detection for 68Ga-FAPI-46 Versus 18F-FDG PET/CT and Conventional CT in Patients with Cholangiocarcinoma.

Management of cholangiocarcinoma is among other factors critically determined by accurate staging. Here, we aimed to assess the accuracy of PET/CT with the novel cancer fibroblast-directed 68Ga-fibroblast activation protein (FAP) inhibitor (FAPI)-46 tracer for cholangiocarcinoma staging and management guidance. Methods: Patients with cholangiocarcinoma from a prospective observational trial were analyzed. 68Ga-FAPI-46 PET/CT detection efficacy was compared with 18F-FDG PET/CT and conventional CT. SUVmax/tumor-to-background ratio (Wilcoxon test) and separately uptake for tumor grade and location (Mann-Whitney U test) were compared. Immunohistochemical FAP and glucose transporter 1 (GLUT1) expression of stromal and cancer cells was analyzed. The impact on therapy management was investigated by pre- and post-PET/CT questionnaires sent to the treating physicians. Results: In total, 10 patients (6 with intrahepatic cholangiocarcinoma and 4 with extrahepatic cholangiocarcinoma; 6 with grade 2 tumor and 4 with grade 3 tumor) underwent 68Ga-FAPI-46 PET/CT and conventional CT; 9 patients underwent additional 18F-FDG PET/CT. Immunohistochemical analysis was performed on the entire central tumor plain in 6 patients. Completed questionnaires were returned in 8 cases. Detection rates for 68Ga-FAPI-46 PET/CT, 18F-FDG PET/CT, and CT were 5, 5, and 5, respectively, for primary tumor; 11, 10, and 3, respectively, for lymph nodes; and 6, 4, and 2, respectively, for distant metastases. 68Ga-FAPI-46 versus 18F-FDG PET/CT SUVmax for primary tumor, lymph nodes, and distant metastases was 14.5 versus 5.2 (P = 0.043), 4.7 versus 6.7 (P = 0.05), and 9.5 versus 5.3 (P = 0.046), respectively, and tumor-to-background ratio (liver) was 12.1 versus 1.9 (P = 0.043) for primary tumor. Grade 3 tumors demonstrated a significantly higher 68Ga-FAPI-46 uptake than grade 2 tumors (SUVmax, 12.6 vs. 6.4; P = 0.009). Immunohistochemical FAP expression was high on tumor stroma (∼90% of cells positive), whereas GLUT1 expression was high on tumor cells (∼80% of cells positive). Overall, average expression intensity was estimated as grade 3 for FAP and grade 2 for GLUT1. Positive 68Ga-FAPI-46 PET findings led to a consequent biopsy workup and diagnosis of cholangiocarcinoma in 1 patient. However, patient treatment was not adjusted on the basis of 68Ga-FAPI-46 PET. Conclusion: 68Ga-FAPI-46 demonstrated superior radiotracer uptake, especially in grade 3 tumors, and lesion detection in patients with cholangiocarcinoma. In line with this result, immunohistochemistry demonstrated high FAP expression on tumor stroma. Accuracy is under investigation in an ongoing investigator-initiated trial.

Fibroblast Activation Protein Inhibitor Theranostics: The Case for Use in Sarcoma.

The theranostic use of fibroblast activation protein inhibitors (FAPIs) is a novel approach in oncology. Sarcomas are a heterogenous group of rare malignant tumors. Prognosis remains poor in advanced/metastatic disease due to limited therapeutic options. Sarcoma frequently demonstrate high expression of fibroblast activation protein alpha on the tumor cells themselves, in contrast to other solid tumors, where it is mainly expressed on cancer-associated fibroblasts. Consequently, high in vivo uptake of FAPI in PET is observed in sarcoma. Moreover, retrospective case reports and series demonstrated feasibility of FAPI radioligand therapy with signs of tumor response.

Functional noninvasive detection of glycolytic pancreatic ductal adenocarcinoma.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) lacks effective treatment options beyond chemotherapy. Although molecular subtypes such as classical and QM (quasi-mesenchymal)/basal-like with transcriptome-based distinct signatures have been identified, deduced therapeutic strategies and targets remain elusive. Gene expression data show enrichment of glycolytic genes in the more aggressive and therapy-resistant QM subtype. However, whether the glycolytic transcripts are translated into functional glycolysis that could further be explored for metabolic targeting in QM subtype is still not known. METHODS: We used different patient-derived PDAC model systems (conventional and primary patient-derived cells, patient-derived xenografts (PDX), and patient samples) and performed transcriptional and functional metabolic analysis. These included RNAseq and Illumina HT12 bead array, in vitro Seahorse metabolic flux assays and metabolic drug targeting, and in vivo hyperpolarized [1-13C]pyruvate and [1-13C]lactate magnetic resonance spectroscopy (HP-MRS) in PDAC xenografts. RESULTS: We found that glycolytic metabolic dependencies are not unambiguously functionally exposed in all QM PDACs. Metabolic analysis demonstrated functional metabolic heterogeneity in patient-derived primary cells and less so in conventional cell lines independent of molecular subtype. Importantly, we observed that the glycolytic product lactate is actively imported into the PDAC cells and used in mitochondrial oxidation in both classical and QM PDAC cells, although more actively in the QM cell lines. By using HP-MRS, we were able to noninvasively identify highly glycolytic PDAC xenografts by detecting the last glycolytic enzymatic step and prominent intra-tumoral [1-13C]pyruvate and [1-13C]lactate interconversion in vivo. CONCLUSION: Our study adds functional metabolic phenotyping to transcriptome-based analysis and proposes a functional approach to identify highly glycolytic PDACs as candidates for antimetabolic therapeutic avenues.

Progranulin mediates immune evasion of pancreatic ductal adenocarcinoma through regulation of MHCI expression.

Immune evasion is indispensable for cancer initiation and progression, although its underlying mechanisms in pancreatic ductal adenocarcinoma (PDAC) are not fully known. Here, we characterize the function of tumor-derived PGRN in promoting immune evasion in primary PDAC. Tumor- but not macrophage-derived PGRN is associated with poor overall survival in PDAC. Multiplex immunohistochemistry shows low MHC class I (MHCI) expression and lack of CD8+ T cell infiltration in PGRN-high tumors. Inhibition of PGRN abrogates autophagy-dependent MHCI degradation and restores MHCI expression on PDAC cells. Antibody-based blockade of PGRN in a PDAC mouse model remarkably decelerates tumor initiation and progression. Notably, tumors expressing LCMV-gp33 as a model antigen are sensitized to gp33-TCR transgenic T cell-mediated cytotoxicity upon PGRN blockade. Overall, our study shows a crucial function of tumor-derived PGRN in regulating immunogenicity of primary PDAC.

Antitumor immune response is associated with favorable survival in GEP-NEN G3.

The tumor immune microenvironment (TME) represents a key determinant for responses to cancer treatment. However, the immune phenotype of highly proliferative gastroenteropancreatic neuroendocrine neoplasms (GEP-NEN) is still largely elusive. In this retrospective study, we characterized the TME of high-grade (G3, Ki-67 > 20%) GEP-NEN. We analyzed formalin-fixed paraffin-embedded samples from 37 patients with GEP-NEN G3 by immunohistochemistry and multiplex immunofluorescence to address the abundance and spatial interaction of relevant immune subsets. We focused on the expression of immune checkpoint molecules PD-1 and PD-L1, the cytotoxic T-cell marker CD8, and the tumor-associated macrophage marker CD206. Findings were correlated with overall survival (OS) from the date of a cancer diagnosis. Patients with PD-L1-positive tumors (CPS ≥ 1) and intense PD-1+CD8+ immune cell infiltration showed the most favorable median OS. Multiplex immunofluorescence staining of ten representative tissue samples illustrated intratumoral heterogeneity of PD-L1 expression. Dense PD-1+CD8+ immune cell infiltrates were observed in PD-L1-positive tumor regions but not in PD-L1-negative regions. Proximity analysis revealed a spatial interaction between PD-1+CD8+ cells and PD-L1-positive cells. Our data suggest a pre-existing antitumor immune response in the TME in a subgroup of GEP-NEN G3. This supports a targeted clinical exploration of immunotherapeutic approaches.

Characterization of a dual BET/HDAC inhibitor for treatment of pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is resistant to virtually all chemo- and targeted therapeutic approaches. Epigenetic regulators represent a novel class of drug targets. Among them, BET and HDAC proteins are central regulators of chromatin structure and transcription, and preclinical evidence suggests effectiveness of combined BET and HDAC inhibition in PDAC. Here, we describe that TW9, a newly generated adduct of the BET inhibitor (+)-JQ1 and class I HDAC inhibitor CI994, is a potent dual inhibitor simultaneously targeting BET and HDAC proteins. TW9 has a similar affinity to BRD4 bromodomains as (+)-JQ1 and shares a conserved binding mode, but is significantly more active in inhibiting HDAC1 compared to the parental HDAC inhibitor CI994. TW9 was more potent in inhibiting tumor cell proliferation compared to (+)-JQ1, CI994 alone or combined treatment of both inhibitors. Sequential administration of gemcitabine and TW9 showed additional synergistic antitumor effects. Microarray analysis revealed that dysregulation of a FOSL1-directed transcriptional program contributed to the antitumor effects of TW9. Our results demonstrate the potential of a dual chromatin-targeting strategy in the treatment of PDAC and provide a rationale for further development of multitarget inhibitors.

Methods to Analyze the Role of Progranulin (PGRN/GEP) on Cancer Stem Cell Features.

Emerging evidence suggests that tumors are hierarchically organized with a distinct subpopulation called cancer stem cells (CSCs) lying at the apex of the hierarchy. These cells are not only responsible for tumor initiation and progression but also endowed with stem cell properties, including self-renewal, chemoresistance, and tumor initiation. Although existing therapies can initially eliminate the bulk population of tumor, the stem cell properties of CSCs enable them to survive and repopulate the tumor, resulting in disease relapse. Recently, our group has shown that progranulin (PGRN/GEP) defined a hepatic cancer stem cell subpopulation in hepatocellular carcinoma. This subpopulation demonstrated enhanced ability for colony and spheroid formation, chemoresistance, and tumor initiation. In this chapter, we describe the methods used to isolate the progranulin+ subpopulation and analyze their CSC properties.

Mouse Monoclonal Antibodies Against Progranulin (PGRN/GEP) as Therapeutics in Preclinical Cancer Models.

The use of monoclonal antibody (mAb) has become a unique means of targeted therapy for human cancers. mAb-based therapies have shown survival benefits by applying alone or in combination with chemotherapeutics. Being a humanized biomolecule with exquisite target specificity, mAb demonstrated effects in a relatively lower dose range with limited off-target harm to the patients. Nowadays, novel targets involved in tumorigenic mechanisms and biomarkers expressed exclusively on cancer cell surface are being constantly discovered. The potential effects of their specific mAb could be investigated in the preclinical cancer model. In this chapter, we outlined our experimental procedures in determining the feasibility of novel mAb in the preclinical cancer model, with an example of progranulin (PGRN/GEP) mAb against hepatocellular carcinoma (HCC) tumor in mouse model. This chapter included the establishment of subcutaneous and orthotopic HCC tumor in mouse model, the injection of the mouse monoclonal antibody in combination with cytotoxic chemotherapeutics, the assessment of tumor development, and the analyses of the molecular changes of the tumor cells.

Notch-Induced Myeloid Reprogramming in Spontaneous Pancreatic Ductal Adenocarcinoma by Dual Genetic Targeting.

Despite advances in our understanding of the genetics of pancreatic ductal adenocarcinoma (PDAC), the efficacy of therapeutic regimens targeting aberrant signaling pathways remains highly limited. Therapeutic strategies are greatly hampered by the extensive desmoplasia that comprises heterogeneous cell populations. Notch signaling is a contentious pathway exerting opposite roles in tumorigenesis depending on cellular context. Advanced model systems are needed to gain more insights into complex signaling in the multilayered tumor microenvironment. In this study, we employed a dual recombinase-based in vivo strategy to modulate Notch signaling specifically in myeloid cells to dissect the tumorigenic role of Notch in PDAC stroma. Pancreas-specific KrasG12D activation and loss of Tp53 was induced using a Pdx1-Flp transgene, whereas Notch signaling was genetically targeted using a myeloid-targeting Lyz2-Cre strain for either activation of Notch2-IC or deletion of Rbpj. Myeloid-specific Notch activation significantly decreased tumor infiltration by protumorigenic M2 macrophages in spontaneous endogenous PDAC, which translated into significant survival benefit. Further characterization revealed upregulated antigen presentation and cytotoxic T effector phenotype upon Notch-induced M2 reduction. This approach is the first proof of concept for genetic targeting and reprogramming of myeloid cells in a complex disease model of PDAC and provides evidence for a regulatory role of Notch signaling in intratumoral immune phenotypes.Significance: This study provides insight into the role of myeloid-dependent NOTCH signaling in PDAC and accentuates the need to dissect differential roles of signaling pathways in different cellular components within the tumor microenvironment. Cancer Res; 78(17); 4997-5010. ©2018 AACR.

Immunotherapy and Combination Strategies in Pancreatic Cancer: Current Status and Emerging Trends.

Pancreatic cancer is among the most aggressive malignancies with no effective therapeutic options thus far. Immunotherapy has recently emerged as a promising alternative for the treatment of various solid tumors. In particular, promising results in clinical trials were observed for therapies targeting immune checkpoint molecules. Efforts have been put into investigating the potential of immunotherapy in treating pancreatic cancer. While most of the clinical trial results are still being awaited, several intrinsic features of pancreatic cancer such as low mutational load and the presence of highly immunosuppressive desmoplasia significantly hamper the efficacy of immunotherapy in this disease. These unique features of pancreatic cancer, however, have advanced our understanding of tumor immunology and might help to tailor the future direction of immunotherapy. In this review, we summarize the current immunotherapeutic strategies and clinical trials targeting checkpoint molecules in pancreatic cancer. Emerging trends towards various combinations with therapies targeting immunosuppressive myeloid cells are also discussed.

Granulin-epithelin precursor interacts with 78-kDa glucose-regulated protein in hepatocellular carcinoma.

BACKGROUND: Granulin-epithelin precursor (GEP) is a secretory growth factor, which has been demonstrated to control cancer growth, invasion, drug resistance and immune escape. Our previous studies and others also demonstrated its potential in targeted therapy. Comprehensive characterization of GEP partner on cancer cells are warranted. We have previously shown that GEP interacted with heparan sulfate on the surface of liver cancer cells and the interaction is crucial for GEP-mediated signaling transduction. This study aims to characterize GEP protein partner at the cell membrane with the co-immunoprecipitation and mass spectrometry approach. METHODS: The membrane fraction from liver cancer model Hep3B was used for capturing binding partner with the specific monoclonal antibody against GEP. The precipitated proteins were analyzed by mass spectrometry. After identifying the GEP binding partner, this specific interaction was validated in additional liver cancer cell line HepG2 by co-immunoprecipitation using GRP78 and GEP antibodies, respectively, as the bait. GRP78 transcript levels in hepatocellular carcinoma (HCC) clinical samples (n = 77 pairs) were examined by real-time quantitative RT-PCR. GEP and GRP78 protein expressions were investigated by immunohistochemistry on paraffin sections. RESULTS: We identified the GEP-binding protein as 78-kDa glucose-regulated protein (GRP78, also named heat shock 70-kDa protein 5, HSPA5). This interaction was validated in independent HCC cell lines. Increased GRP78 mRNA levels were demonstrated in liver cancer tissues compared with the paralleled liver tissues (t-test, P = 0.002). GRP78 and GEP transcript levels were significantly correlated (Spearman's correlation, P = 0.001), and the proteins were also detectable in the cytoplasm of liver cancer cells by immunohistochemical staining. CONCLUSIONS: GRP78 and GEP are interacting protein partners in liver cancer cells and may play a role in GEP-mediated cancer progression in HCC.

Comprehensive characterization of the patient-derived xenograft and the paralleled primary hepatocellular carcinoma cell line.

BACKGROUND: Hepatocellular carcinoma (HCC) is an aggressive cancer with high mortality and morbidity worldwide. The limited clinically relevant model has impeded the development of effective HCC treatment strategy. Patient-derived xenograft (PDX) models retain most of the characteristics of original tumors and were shown to be highly predictive for clinical outcomes. Notably, primary cell line models allow in-depth molecular characterization and high-throughput analysis. Combined usage of the two models would provide an excellent tool for systematic study of therapeutic strategies. Here, we comprehensively characterized the novel PDX and the paralleled primary HCC cell line model. METHODS: Tumor tissues were collected from HCC surgical specimens. HCC cells were sorted for in vivo PDX and in vitro cell line establishment by the expression of hepatic cancer stem cell marker to enhance cell viability and the rate of success on subsequent culture. The PDX and its matching primary cell line were authenticated and characterized in vitro and in vivo. RESULTS: Among the successful cases for generating PDXs and primary cells, HCC40 is capable for both PDX and primary cell line establishment, which were then further characterized. The novel HCC40-PDX and HCC40-CL exhibited consistent phenotypic characteristics as the original tumor in terms of HBV protein and AFP expressions. In common with HCC40-PDX, HCC40-CL was tumorigenic in immunocompromised mice. The migration ability in vitro and metastatic properties in vivo echoed the clinical feature of venous infiltration. Genetic profiling by short tandem repeat analysis and p53 mutation pattern consolidated that both the HCC40-PDX and HCC40-CL models were derived from the HCC40 clinical specimen. CONCLUSIONS: The paralleled establishment of PDX and primary cell line would serve as useful models in comprehensive studies for HCC pathogenesis and therapeutics development for personalized treatment.

Hepatic cancer stem cell marker granulin-epithelin precursor and ß-catenin expression associate with recurrence in hepatocellular carcinoma.

Granulin-epithelin precursor (GEP) has been demonstrated to confer enhanced cancer stem-like cell properties in hepatocellular carcinoma (HCC) cell line models in our previous studies. Here, we aimed to examine the GEP-expressing cells in relation to the stem cell related molecules and stem-like cell properties in the prospective HCC clinical cohort. GEP protein levels were significantly higher in HCCs than the paralleled non-tumor liver tissues, and associated with venous infiltration. GEPhigh cells isolated from clinical HCC samples exhibited higher levels of stem cell marker CD133, pluripotency-associated signaling molecules ß-catenin, Oct4, SOX2, Nanog, and chemodrug transporter ABCB5. In addition, GEPhigh cells possessed preferential ability to form colonies and spheroids, and enhanced in vivo tumor-initiating ability while their xenografts were able to be serially subpassaged into secondary mouse recipients. Expression levels of GEP and pluripotency-associated genes were further examined in the retrospective HCC cohort and demonstrated significant correlation of GEP with ß-catenin. Notably, HCC patients with high GEP and ß-catenin levels demonstrated poor recurrence-free survival. In summary, GEP-positive HCC cells directly isolated from clinical specimens showed ß-catenin elevation and cancer stem-like cell properties.

Copy number gain of granulin-epithelin precursor (GEP) at chromosome 17q21 associates with overexpression in human liver cancer.

BACKGROUND: Granulin-epithelin precursor (GEP), a secretory growth factor, demonstrated overexpression in various human cancers, however, mechanism remain elusive. Primary liver cancer, hepatocellular carcinoma (HCC), ranks the second in cancer-related death globally. GEP controlled growth, invasion, metastasis and chemo-resistance in liver cancer. Noted that GEP gene locates at 17q21 and the region has been frequently reported to be amplified in subset of HCC. The study aims to investigate if copy number gain would associate with GEP overexpression. METHODS: Quantitative Microsatellite Analysis (QuMA) was used to quantify the GEP DNA copy number, and fluorescent in situ hybridization (FISH) was performed to consolidate the amplification status. GEP gene copy number, mRNA expression level and clinico-pathological features were analyzed. RESULTS: GEP DNA copy number determined by QuMA corroborated well with the FISH data, and the gene copy number correlated with the expression levels (n = 60, r = 0.331, P = 0.010). Gain of GEP copy number was observed in 20% (12/60) HCC and associated with hepatitis B virus infection status (P = 0.015). In HCC with increased GEP copy number, tight association between GEP DNA and mRNA levels were observed (n = 12, r = 0.664, P = 0.019). CONCLUSIONS: Gain of the GEP gene copy number was observed in 20% HCC and the frequency comparable to literatures reported on the chromosome region 17q. Increased gene copy number contributed to GEP overexpression in subset of HCC.

Granulin-epithelin precursor renders hepatocellular carcinoma cells resistant to natural killer cytotoxicity.

Immunoevasion is an emerging hallmark of cancer. Impairment of natural killer (NK) cytotoxicity is a mechanism to evade host immunosurveillance. Granulin-epithelin precursor (GEP) is a hepatic oncofetal protein regulating growth, invasion, and chemoresistance in hepatocellular carcinoma (HCC). We examined the role of GEP in conferring HCC cells the ability to evade NK cytotoxicity. In HCC cell lines, GEP overexpression reduced, whereas GEP suppression enhanced sensitivity to NK cytotoxicity. GEP downregulated surface expression of MHC class I chain-related molecule A (MICA), ligand for NK stimulatory receptor NK group 2 member D (NKG2D), and upregulated human leukocyte antigen-E (HLA-E), ligand for NK inhibitory receptor CD94/NKG2A. Functionally, GEP augmented production of soluble MICA, which suppressed NK activation. Matrix metalloproteinase (MMP)2 and MMP9 activity was involved partly in the GEP-regulated MICA shedding from HCC cells. In primary HCCs (n = 80), elevated GEP (P < 0.001), MICA (P < 0.001), and HLA-E (P = 0.089) expression was observed when compared with those in nontumor (n = 80) and normal livers (n = 10). Serum GEP (P = 0.010) and MICA (P < 0.001) levels were higher in patients with HCC (n = 80) than in healthy individuals (n = 30). High serum GEP and/or MICA levels were associated with poor recurrence-free survival (log-rank test, P = 0.042). Importantly, GEP blockade by mAbs sensitized HCC cells to NK cytotoxicity through MICA. In summary, GEP rendered HCC cells resistant to NK cytotoxicity by modulating MICA expression, which could be reversed by GEP blockade using antibody. Serum GEP and MICA levels are prognostic factors and can be used to stratify patients for targeted therapy.

Antibody against granulin-epithelin precursor sensitizes hepatocellular carcinoma to chemotherapeutic agents.

Granulin-epithelin precursor (GEP) overexpression has been shown in many cancers with functional role on growth, and recently on regulating chemoresistance and cancer stem cell (CSC) properties. Here, we investigate the combined effect of GEP antibody and chemotherapeutic agent. Combination therapy was compared with monotherapy using hepatocellular carcinoma (HCC) cells in vitro and orthotopic liver tumor models in vivo. CD133 and related hepatic CSC marker expressions were investigated by flow cytometry. Antiproliferative and apoptotic effects and signaling mechanisms were examined by immunohistochemistry, flow cytometry, and Western blot analysis. Secretory GEP levels in the serum and culture supernatant samples were measured by ELISA. We demonstrated that HCC cells that survived under chemotherapeutic agents showed upregulation of hepatic CSC markers CD133/GEP/ABCB5, and enhanced colony and spheroid formation abilities. Importantly, GEP antibody sensitized HCC cells to the apoptosis induced by chemotherapy for both HCC cell lines and the chemoresistant subpopulations, and counteracted the chemotherapy-induced GEP/ABCB5 expressions and Akt/Bcl-2 signaling. In human HCC orthotopic xenograft models, GEP antibody treatment alone was consistently capable of inhibiting the tumor growth. Notably, combination of GEP antibody with high dose of cisplatin resulted in the eradication of all established intrahepatic tumor in three weeks. This preclinical study demonstrated that GEP antibody sensitized HCC cells to apoptosis induced by chemotherapeutic agents. Combination treatment with GEP antibody and chemotherapeutic agent has the potential to be an effective therapeutic regimen for GEP-expressing cancers.