Fibrolamellar hepatocellular carcinoma (FL-HCC) has historically been classified as a rare subtyp... more Fibrolamellar hepatocellular carcinoma (FL-HCC) has historically been classified as a rare subtype of HCC. However, unlike "classic" HCC, it occurs in children and young adults without underlying liver disease. The recent discovery of a deletion mutation in all FL-HCCs represented a major advancement in understanding the pathogenesis of this disease. This deletion results in the fusion of the genes encoding a heat shock protein (DNAJB1) and the catalytic subunit of protein kinase A (PKA, PRKACA), and overexpression of PRKACA and enhanced cAMP-dependent PKA activity. This review summarizes recent advancements in FL-HCC pathogenesis and characteristics of the HSP40-PKA C protein.
Angiomyolipomata (AML) belong to a family of tumors known as perivascular epithelioid cell tumors... more Angiomyolipomata (AML) belong to a family of tumors known as perivascular epithelioid cell tumors (PEComas) that share a common immunophenotypic profile of muscle and melanocytic differentiation. These tumors are clonal in nature and have a strong association with tuberous sclerosis. Genetic analyses have reported allelic imbalance at the TSC2 locus on 16p13. In the context of non-TSC, non-LAM associated AMLs and non-renal PEComas, the functional status of the TSC2 signaling pathway has not been reported. Studies over the last several years have uncovered a critical role of the TSC1/2 genes in negatively regulating the Rheb/mTOR/p70S6K cascade. Here, we examined the activity of this pathway in sporadic AMLs and PEComas using immunohistochemical and biochemical analyses. We found increased levels of phospho-p70S6K, a marker of mTOR activity, in 15 of 15 non-TSC AMLs. This was accompanied by reduced phospho-AKT expression, a pattern that is consistent with the disruption of TSC1/2 function. Western blot analysis confirmed mTOR activation concurrent with the loss of TSC2 and not TSC1 in sporadic AMLs. Similarly, elevated phospho-p70S6K and reduced phospho-AKT expression was detected in 14/15 cases of extra-renal PEComas. These observations provide the first functional evidence that mTOR activation is common to sporadic, non-TSC-related AMLs and PEComas. This suggests the possibility that mTOR inhibitors such as rapamycin may be therapeutic for this class of disease.
Journal of the National Cancer Institute, Mar 18, 1998
Background: The fragile histidine triad (FHIT) gene at chromosome 3p14.2 has been proposed to be ... more Background: The fragile histidine triad (FHIT) gene at chromosome 3p14.2 has been proposed to be a candidate tumor suppressor gene in human cancers. To test whether FHIT exhibits the functional properties of a tumor suppressor gene, we studied the expression of its protein (pFHIT) in human carcinoma cells and examined the ability of FHIT to inhibit the neoplastic phenotype of cancer cells. Methods: Subcellular localization and patterns of protein expression in tumor cells were determined by immunohistochemical analysis and immunoblotting with the use of polyclonal anti-pFHIT antisera. In tumor cells with undetectable pFHIT, we examined the effect of recombinant pFHIT expression on morphology, growth rate, colony formation, and in vivo tumor formation. Results: We demonstrated that pFHIT is a cytoplasmic 17-kd polypeptide whose expression could not be detected in 30 of 52 human carcinoma cell lines tested. We observed, however, that the stable overexpression of pFHIT did not alter cell morphology, inhibit colony formation, or inhibit cell proliferation in vitro. Furthermore, overexpression of pFHIT did not lead to altered cell cycle kinetics in dividing cells. The in vivo tumorigenicity of a tumor cell line that expressed high levels of recombinant pFHIT was equivalent to that of control transfectants and of parental cells. Conclusions: These results suggest that the replacement of pFHIT in human carcinoma cells does not suppress tumor cell growth and that this protein may be involved in tumorigenesis in ways that are distinct from the ''classic'' tumor suppressor paradigm. [
Despite advances in interventional procedures and chemotherapeutic drug development, hepatocellul... more Despite advances in interventional procedures and chemotherapeutic drug development, hepatocellular carcinoma (HCC) is still the fourth leading cause of cancer-related deaths worldwide with a <30% 5-year survival rate. This poor prognosis can be attributed to the fact that HCC most commonly occurs in patients with pre-existing liver conditions, rendering many treatment options too aggressive. Patient survival rates could be improved by a more targeted approach. Ultrasound-induced cavitation can provide a means for overcoming traditional barriers defining drug uptake. The goal of this work was to evaluate preclinical efficacy of image-guided, cavitation-enabled drug delivery with a clinical ultrasound scanner. To this end, ultrasound conditions (unique from those used in imaging) were designed and implemented on a Philips EPIQ and S5-1 phased array probe to produced focused ultrasound for cavitation treatment. Sonovue ® microbubbles which are clinically approved as an ultrasound contrast agent were used for both imaging and cavitation treatment. A genetically engineered mouse model was bred and used as a physiologically relevant preclinical analog to human HCC. It was observed that imageguided and targeted microbubble cavitation resulted in selective disruption of the tumor blood flow and enhanced doxorubicin uptake and penetration. Histology results indicate that no gross morphological damage occurred as a result of this process. The combination of these effects may be exploited to treat HCC and other challenging malignancies and could be implemented with currently available ultrasound scanners and reagents.
Background: The potential health effects of polybrominated diphenyl ethers (PBDEs) that are widel... more Background: The potential health effects of polybrominated diphenyl ethers (PBDEs) that are widely used as flame-retardants in consumer products have been attributed, in part, to their endocrine disrupting properties. The purpose of this study is to examine the in vivo effects of an early exposure to PBDEs on the development of insulin resistance in mice. Results: The metabolic consequences of BDE-47 in mice with varying insulin sensitivities secondary to liver-specific activation of Akt (Pten fl/fl ;Alb Cre) and mTORC1 (Tsc1 fl/fl ;Alb Cre) as well as wild-type littermates, were studied. BDE-47, a dominant congener of PBDE, was given daily (1 mg/kg/day) for six weeks by oral gavage in young mice following weaning. At the end of the exposure, there were no significant differences in total body, liver, or white adipose tissue weights between the BDE-47-treated vs. DMSO-treated mice for each respective genotype. Metabolic studies revealed significant impairment in insulin sensitivity in the BDE-47-treated Pten fl/fl ;Alb Cre mice, but not in wild-type or Tsc1 fl/fl ;Alb Cre mice. This was not accompanied by significant alterations in plasma insulin levels or hepatic triglyceride accumulation in the Pten fl/fl ;Alb Cre mice. The mean plasma BDE-47 level in the wild-type mice was 11.7 ± 2.9 ng/g (wet weight). Conclusions: Our findings indicate that BDE-47 exposure during the early post-natal period induces a mild disturbance in glucose metabolism in susceptible mice with increased baseline insulin sensitivity. These results suggest an interaction between BDE-47 and genetic factors that regulate insulin signaling, which may result in long-term consequences.
With the widespread adoption of molecular profiling in clinical oncology practice, many physician... more With the widespread adoption of molecular profiling in clinical oncology practice, many physicians are faced with making therapeutic decisions based upon isolated genomic alterations. For example, epidermal growth factor receptor tyrosine kinase inhibitors (TKIs) are effective in EGFR-mutant non-small cell lung cancers (NSCLC) while anti-EGFR monoclonal antibodies are ineffective in Rasmutant colorectal cancers. The matching of mutations with drugs aimed at their respective gene products represents the current state of "precision" oncology. Despite the great expectations of this approach, only a fraction of cancers responds to 'targeted' interventions, and many early responders will ultimately develop resistance to these agents. The underwhelming success of mutation-driven therapies across all cancer types is not due to an inability to detect genetic changes in tumors; rather a deficit in functional insight into the genomic alterations that give rise to each cancer. The Achilles heel of precision oncology thus remains the lack of a robust functional understanding of an individual cancer genome that then allows prediction of the best therapy and resultant outcome for that patient. Current practice focuses on one 'actionable' mutation at a time, while solid cancers typically possess many mutations that involve different cellular sub-populations within a tumor. No method or platform currently exists to guide the interpretation of these complex data, nor to accurately predict response to treatment. This problem is particularly germane to primary liver cancers (PLC), for which only a handful of targeted therapies have been introduced. Here, we will review strategies aimed at overcoming some of these challenges in precision oncology, using liver cancer as an example.
Insulin resistance is an important contributing factor in non-alcoholic fatty liver disease. AKT ... more Insulin resistance is an important contributing factor in non-alcoholic fatty liver disease. AKT and mTORC1 are key components of the insulin pathway, and play a role in promoting de novo lipogenesis. However, mTORC1 hyperactivity per se does not induce steatosis in mouse livers, but instead, protects against high-fat diet induced steatosis. Here, we investigate the in vivo mechanism of steatosis-resistance secondary to mTORC1 activation, with emphasis on the role of S6K1-mediated feedback inhibition of AKT. Mice with single or double deletion of Tsc1 and/or S6k1 in a liver-specific or whole-body manner were generated to study glucose and hepatic lipid metabolism between the ages of 6-14 weeks. Following 8 weeks of high-fat diet, the Tsc1-/-;S6k1-/-mice had lower body weights but higher liver TG levels compared to that of the Tsc1-/-mice. However, the loss of S6k1 did not relieve feedback inhibition of Akt activity in the Tsc1-/-livers. To overcome Akt suppression, Pten was deleted in Tsc1-/-livers, and the resultant mice showed improved glucose tolerance compared with the Tsc1-/-mice. However, liver TG levels were significantly reduced in the Tsc1-/-;Pten-/-mice compared to the Pten-/-mice, which was restored with rapamycin. We found no correlation between liver TG and serum NEFA levels. Expression of lipogenic genes (Srebp1c, Fasn) were elevated in the Tsc1-/-;Pten-/-livers, but this was counterbalanced by an up-regulation of Cpt1a involved in fatty acid oxidation and the anti-oxidant protein, Nrf2. In summary, our in vivo models showed that mTORC1-induced resistance to steatosis was dependent on S6K1 activity, but not secondary to AKT suppression. These findings confirm that AKT and mTORC1 have opposing effects on hepatic lipid metabolism in vivo.
Proceedings of the National Academy of Sciences of the United States of America, Dec 22, 1998
Tuberous sclerosis is an autosomal dominant disorder characterized by the development of aberrant... more Tuberous sclerosis is an autosomal dominant disorder characterized by the development of aberrant growths in many tissues and organs. Linkage analysis revealed two disease-determining genes on chromosome 9 and chromosome 16. The tuberous sclerosis complex gene-2 (TSC2) on chromosome 16 encodes the tumor suppressor protein tuberin. We have shown earlier that loss of TSC2 is sufficient to induce quiescent cells to enter the cell cycle. Here we show that TSC2-negative fibroblasts exhibit a shortened G 1 phase. Although the expression of cyclin E, cyclin A, p21, or Cdc25A is unaffected, TSC2-negative cells express much lower amounts of the cyclin-dependent kinase (CDK) inhibitor p27 because of decreased protein stability. In TSC2 mutant cells the amount of p27 bound to CDK2 is diminished, accompanied with elevated kinase activity. Ectopic expression studies revealed that the aforementioned effects can be reverted by transfecting TSC2 in TSC2-negative cells. High ectopic levels of p27 have cell cycle inhibitory effects in TSC2-positive cells but not in TSC2-negative counterparts, although the latter still depend on CDK2 activity. Loss of TSC2 induces soft agar growth of fibroblasts, a process that cannot be inhibited by high levels of p27. Both phenotypes of TSC2-negative cells, their resistance to the activity of ectopic p27, and the instability of endogenous p27, could be explained by our observation that the nucleoprotein p27 is mislocated into the cytoplasm upon loss of TSC2. These findings provide insights into the molecular mechanism of how loss of TSC2 induces cell cycle entry and allow a better understanding of its tumor suppressor function. MATERIALS AND METHODS Cells, Cell Culture, Flow Cytometry, and Centrifugal Elutriation. EEF4 (TSC2-positive) and EEF8 (TSC2-negative) cells were derived from Eker rat embryos homozygous for the wild-type and the Eker-mutant TSC2 gene, respectively. Whole embryos were removed on day 10.5 before in utero deaths of the Eker homozygous mutants have occurred. Sam-The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ''advertisement'' in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Organotypic tumor slices represent a physiologically-relevant culture system for studying the tum... more Organotypic tumor slices represent a physiologically-relevant culture system for studying the tumor microenvironment. Systematic characterization of the tumor slice culture system will enable its effective application for translational research. Here, using flow cytometry-based immunophenotyping, we performed a comprehensive characterization of the immune cell composition in organotypic tumor slices prepared from four syngeneic mouse tumor models and a human liver tumor. We found that the immune cell compositions of organotypic tumor slices prepared on the same day as the tumor cores were harvested are similar. Differences were primarily observed in the lymphocyte population of a clinical hepatocellular carcinoma case. Viable populations of immune cells persisted in the tumor slices for 7 days. Despite some changes in the immune cell populations, we showed the utility of mouse tumor slices for assessing responses to immune-modulatory agents. Further, we demonstrated the ability to use patient-derived xenograft tumor slices for assessing responses to targeted and cytotoxic drugs. Overall, tumor slices provide a broadly useful platform for studying the tumor microenvironment and evaluating the preclinical efficacy of cancer therapeutics.
Objective Programmed cell death protein 1 (PD-1) checkpoint inhibition and adoptive cellular ther... more Objective Programmed cell death protein 1 (PD-1) checkpoint inhibition and adoptive cellular therapy have had limited success in patients with microsatellite stable colorectal cancer liver metastases (CRLM). We sought to evaluate the effect of interleukin 10 (IL-10) blockade on endogenous T cell and chimeric antigen receptor T (CAR-T) cell antitumour function in CRLM slice cultures. Design We created organotypic slice cultures from human CRLM (n=38 patients' tumours) and tested the antitumour effects of a neutralising antibody against IL-10 (αIL-10) both alone as treatment and in combination with exogenously administered carcinoembryonic antigen (CEA)-specific CART cells. We evaluated slice cultures with single and multiplex immunohistochemistry, in situ hybridisation, single-cell RNA sequencing, reverse-phase protein arrays and time-lapse fluorescent microscopy. Results αIL-10 generated a 1.8-fold increase in T cell-mediated carcinoma cell death in human CRLM slice cultures. αIL-10 significantly increased proportions of CD8 + T cells without exhaustion transcription changes, and increased human leukocyte antigen-DR isotype (HLA-DR) expression of macrophages. The antitumour effects of αIL-10 were reversed by major histocompatibility complex class I or II (MHC-I or MHC-II) blockade, confirming the essential role of antigen presenting cells. Interrupting IL-10 signalling also rescued murine CART cell proliferation and cytotoxicity from myeloid cell-mediated immunosuppression. In human CRLM slices, αIL-10 increased CEA-specific CART cell activation and CART cell-mediated cytotoxicity, with nearly 70% carcinoma cell apoptosis across multiple human tumours. Pretreatment with an IL-10 receptor blocking antibody also potentiated CART function. Conclusion Neutralising the effects of IL-10 in human CRLM has therapeutic potential as a stand-alone treatment and to augment the function of adoptively transferred CART cells. HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY ⇒ IL-10 represents a major immunosuppressive signal to overcome in patients with CRLM. Targeting IL-10/IL-10 receptor signalling has great potential to be incorporated into systemic therapy for patients with immune checkpoint blockade-resistant, MSS CRLM. CART cells, armed against IL-10 suppression, may pave the way for more effective adoptive cell therapies for patients with MSS CRLM refractory to currently available surgical and medical therapies.
ABSTRACTThe DNAJ-PKAc fusion kinase is a defining feature of the adolescent liver cancer fibrolam... more ABSTRACTThe DNAJ-PKAc fusion kinase is a defining feature of the adolescent liver cancer fibrolamellar carcinoma (FLC). A single lesion on chromosome 19 generates this mutant kinase by creating a fused gene encoding the chaperonin binding domain of Hsp40 (DNAJ) in frame with the catalytic core of protein kinase A (PKAc). FLC tumors are notoriously resistant to standard chemotherapies. Aberrant kinase activity is assumed to be a contributing factor. Yet recruitment of binding partners, such as the chaperone Hsp70, implies that the scaffolding function of DNAJ- PKAc may also underlie pathogenesis. By combining proximity proteomics with biochemical analyses and photoactivation live-cell imaging we demonstrate that DNAJ-PKAc is not constrained by A-kinase anchoring proteins. Consequently, the fusion kinase phosphorylates a unique array of substrates. One validated DNAJ-PKAc target is the Bcl-2 associated athanogene 2 (BAG2), a co-chaperone recruited to the fusion kinase through associat...
Hepatocellular carcinoma (HCC) is a significant cause of morbidity and mortality worldwide with l... more Hepatocellular carcinoma (HCC) is a significant cause of morbidity and mortality worldwide with limited therapeutic options for advanced disease. Targeted alpha therapy (TAT) is an emerging class of targeted cancer therapy in which alpha-particle-emitting radionuclides, such as thorium-227, are specifically delivered to cancer tissue. Glypican-3 (GPC3) is a cell surface glycoprotein highly expressed on HCC. In this study, we describe the development and in vivo efficacy of a 227 Th-labeled GPC3 targeting antibody conjugate (227 Th-octapa-αGPC3) for treatment of HCC in an orthotopic murine model. METHODS The chelator p-SCN-Bn-H4octapa-NCS (octapa) was conjugated to a GPC3 targeting antibody (αGPC3) for subsequent 227 Th radiolabeling (octapa-αGPC3). Conditions were varied to optimize radiolabeling of 227 Th. In vitro stability was evaluated by measuring percentage of protein-bound 227 Th by gamma-ray spectroscopy. An orthotopic athymic Nu/J murine model using HepG2 cells was developed. Biodistribution and blood clearance of 227 Th-octapa-αGPC3 was evaluated in tumor bearing mice. Efficacy of 227 Th-octapa-αGPC3 was assessed in tumor bearing animals with serial measurement of serum alpha-fetoprotein at 23 days after radionuclide injection. RESULTS Octapa-conjugated αGPC3 provided up to 70% 227 Th labeling yield in 2 h at room temperature. In the presence of ascorbate, 97.8% of 227 Th was bound to αGPC3-octapa after 14 d in phosphate buffered saline. In HepG2 tumor-bearing mice, highly specific GPC3 targeting was observed, with significant 227 Th-octapa-αGPC3 accumulation in the tumor over time and minimal accumulation in normal tissue. 23 days after treatment, significant reduction in tumor burden was observed in 4 mice receiving 500 kBq/kg 227 Th-octapa-αGPC3 by tail vein injection. No acute off-target toxicity was observed and no animals died prior to termination of the study. CONCLUSION 227 Th-octapa-αGPC3 was observed to be stable in vitro, maintain high specificity for GPC3 with favorable biodistribution in vivo, and result in significant antitumor activity without significant acute off-target toxicity in an orthotopic murine model of HCC.
Glypican-3 (GPC3) is a tumor associated antigen expressed by hepatocellular carcinoma (HCC) cells... more Glypican-3 (GPC3) is a tumor associated antigen expressed by hepatocellular carcinoma (HCC) cells. This preclinical study evaluated the efficacy of a theranostic platform using a GPC3-targeting antibody αGPC3 conjugated to zirconium-89 (89Zr) and yttrium-90 (90Y) to identify, treat, and assess treatment response in a murine model of HCC. A murine orthotopic xenograft model of HCC was generated. Animals were injected with 89Zr-labeled αGPC3 and imaged with a small-animal positron emission/computerized tomography (PET/CT) imaging system (immuno-PET) before and 30 days after radioimmunotherapy (RIT) with 90Y-labeled αGPC3. Serum alpha fetoprotein (AFP), a marker of tumor burden, was measured. Gross tumor volume (GTV) and SUVmax by immuno-PET was measured using fixed intensity threshold and manual segmentation methods. Immuno-PET GTV measurements reliably quantified tumor burden prior to RIT, strongly correlating with serum AFP (R2 = 0.90). Serum AFP was significantly lower 30 days afte...
Direct-Acting Antiviral (DAA) drugs are highly effective in eliminating HCV in most chronically i... more Direct-Acting Antiviral (DAA) drugs are highly effective in eliminating HCV in most chronically infected subjects, but it is unclear whether the multiple mechanisms employed by HCV to disable both innate and adaptive immunity cease to function as soon as HCV is eliminated. To test this, we evaluated the capacity of human liver tissue to respond to TLR3 and TLR4 ligands using non-infected liver tissue, tissue with active HCV infection, and tissue from which HCV had been eliminated by DAA. We found that DAA-treated, formerly HCV-infected liver tissue manifested ongoing abnormalities of innate immunity that mapped to liver non-parenchymal cells (NPC). Hepatic innate immunity was not suppressed but enhanced in HCV-infected tissue, and these abnormalities were not corrected in the successfully DAA-treated liver tissue. In conclusion, ongoing immune activation persists in formerly HCV-infected but now DAA-cured liver.
With the widespread adoption of molecular profiling in clinical oncology practice, many physician... more With the widespread adoption of molecular profiling in clinical oncology practice, many physicians are faced with making therapeutic decisions based upon isolated genomic alterations. For example, epidermal growth factor receptor tyrosine kinase inhibitors (TKIs) are effective in EGFR-mutant non-small cell lung cancers (NSCLC) while anti-EGFR monoclonal antibodies are ineffective in Rasmutant colorectal cancers. The matching of mutations with drugs aimed at their respective gene products represents the current state of "precision" oncology. Despite the great expectations of this approach, only a fraction of cancers responds to 'targeted' interventions, and many early responders will ultimately develop resistance to these agents. The underwhelming success of mutation-driven therapies across all cancer types is not due to an inability to detect genetic changes in tumors; rather a deficit in functional insight into the genomic alterations that give rise to each cancer. The Achilles heel of precision oncology thus remains the lack of a robust functional understanding of an individual cancer genome that then allows prediction of the best therapy and resultant outcome for that patient. Current practice focuses on one 'actionable' mutation at a time, while solid cancers typically possess many mutations that involve different cellular sub-populations within a tumor. No method or platform currently exists to guide the interpretation of these complex data, nor to accurately predict response to treatment. This problem is particularly germane to primary liver cancers (PLC), for which only a handful of targeted therapies have been introduced. Here, we will review strategies aimed at overcoming some of these challenges in precision oncology, using liver cancer as an example.
Organotypic tumor slices represent a physiologically-relevant culture system for studying the tum... more Organotypic tumor slices represent a physiologically-relevant culture system for studying the tumor microenvironment. Systematic characterization of the tumor slice culture system will enable its effective application for translational research. Here, using flow cytometry-based immunophenotyping, we performed a comprehensive characterization of the immune cell composition in organotypic tumor slices prepared from four syngeneic mouse tumor models and a human liver tumor. We found that the immune cell compositions of organotypic tumor slices prepared on the same day as the tumor cores were harvested are similar. Differences were primarily observed in the lymphocyte population of a clinical hepatocellular carcinoma case. Viable populations of immune cells persisted in the tumor slices for 7 days. Despite some changes in the immune cell populations, we showed the utility of mouse tumor slices for assessing responses to immune-modulatory agents. Further, we demonstrated the ability to use patient-derived xenograft tumor slices for assessing responses to targeted and cytotoxic drugs. Overall, tumor slices provide a broadly useful platform for studying the tumor microenvironment and evaluating the preclinical efficacy of cancer therapeutics.
Fibrolamellar carcinoma (FLC) is a rare liver cancer. FLCs uniquely produce DNAJ-PKAc, a chimeric... more Fibrolamellar carcinoma (FLC) is a rare liver cancer. FLCs uniquely produce DNAJ-PKAc, a chimeric enzyme consisting of a chaperonin-binding domain fused to the Cα subunit of protein kinase A. Biochemical analyses of clinical samples reveal that a unique property of this fusion enzyme is the ability to recruit heat shock protein 70 (Hsp70). This cellular chaperonin is frequently up-regulated in cancers. Gene-editing of mouse hepatocytes generated disease-relevant AML12DNAJ-PKAc cell lines. Further analyses indicate that the proto-oncogene A-kinase anchoring protein-Lbc is up-regulated in FLC and functions to cluster DNAJ-PKAc/Hsp70 sub-complexes with a RAF-MEK-ERK kinase module. Drug screening reveals Hsp70 and MEK inhibitor combinations that selectively block proliferation of AML12DNAJ-PKAc cells. Phosphoproteomic profiling demonstrates that DNAJ-PKAc biases the signaling landscape toward ERK activation and engages downstream kinase cascades. Thus, the oncogenic action of DNAJ-PKAc ...
Cellular and Molecular Gastroenterology and Hepatology, 2019
Small RNA profiling of primary fibrolamellar carcinoma tumors shows microRNA-375 (miR-375) as the... more Small RNA profiling of primary fibrolamellar carcinoma tumors shows microRNA-375 (miR-375) as the most dysregulated miRNA. Several disease models recapitulate the dramatic suppression of miR-375. Functional interrogation of miR-375 in a novel fibrolamellar carcinoma cell line shows that it suppresses fibrolamellar carcinoma cell growth and migration. BACKGROUND & AIMS: Fibrolamellar carcinoma (FLC) is a rare liver cancer that primarily affects adolescents and young adults. It is characterized by a heterozygous approximately 400-kb deletion on chromosome 19 that results in a unique fusion between DnaJ heat shock protein family member B1 (DNAJB1) and the alpha catalytic subunit of protein kinase A (PRKACA). The role of microRNAs (miRNAs) in FLC remains unclear. We identified dysregulated miRNAs in FLC and investigated whether dysregulation of 1 key miRNA contributes to FLC pathogenesis. METHODS: We analyzed small RNA sequencing (smRNA-seq) data from The Cancer Genome Atlas to identify dysregulated miRNAs in primary FLC tumors and validated the findings in 3 independent FLC cohorts. smRNA-seq also was performed on a FLC patient-derived xenograft model as well as purified cell populations of the liver to determine whether key miRNA changes were tumor cell-intrinsic. We then used clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (Cas9) technology and transposon-mediated gene transfer in mice to determine if the presence of DNAJB1-PRKACA is sufficient to suppress miR-375 expression. Finally, we established a new FLC cell line and performed colony formation and scratch wound assays to determine the functional consequences of miR-375 overexpression. RESULTS: We identified miR-375 as the most dysregulated miRNA in primary FLC tumors (27-fold down-regulation; P ¼ .009). miR-375 expression also was decreased significantly in a FLC patient-derived xenograft model compared to 4 different cell populations of the liver. Introduction of DNAJB1-PRKACA by clustered regularly interspaced short palindromic repeats/CRISPRassociated protein 9 engineering and transposon-mediated somatic gene transfer in mice was sufficient to induce significant loss of miR-375 expression (P < .05). Overexpression of miR-375 in FLC cells inhibited Hippo signaling pathway proteins, including yes-associated protein 1 and connective tissue growth factor, and suppressed cell proliferation and migration (P < .05). CONCLUSIONS: We identified miR-375 as the most downregulated miRNA in FLC tumors and showed that overexpression of miR-375 mitigated tumor cell growth and invasive potential. These findings open a potentially new molecular therapeutic approach. Further studies are necessary to determine how DNAJB1-PRKACA suppresses miR-375 expression and whether miR-375 has additional important targets in this tumor.
Tuberous sclerosis complex (TSC) presents in the pediatric population with a constellation of ben... more Tuberous sclerosis complex (TSC) presents in the pediatric population with a constellation of benign tumors that affect the brain, heart, kidney, lung, and skin. No therapy has been shown to halt disease progression or to prevent its onset. The pathogenesis of TSC stems from the inactivation of one of the two TSC genes, TSC1 and TSC2. A key function of these genes is to regulate the mammalian target of rapamycin (mTOR) pathway in response to cellular energy and nutrient and growth factor availability. Consequently, TSC-related tumors exhibit uncontrolled activation of mTOR and its effectors. Previous work has shown that a specific mTOR inhibitor, rapamycin, effectively downregulated mTOR activity in renal tumors of Eker rats that carry a germline Tsc2 mutation. Using this model, we investigated the effects of rapamycin on pituitary and renal tumors. We observed that rats with pituitary tumors had significantly shorter survival than those without pituitary pathology. Treatment with rapamycin effectively improved their clinical state and prolonged their survival. Rapamycin also resulted in a significant decrease in the size of the Tsc2-related renal tumors. In both types of pathology, tumor response was accompanied by down-regulation of ribo-somal S6 kinase activity, reduction in cell size, and induction of apoptosis. Evidence for drug resistance was found in a small percentage of lesions after prolonged therapy. When rapamycin was given before onset of disease, subsequent development of macroscopic renal tumors was reduced, but no effect on the number of microscopic precursor lesions was found. We conclude that rapamycin-sensitive mTOR activity was critical to tumor progression in the Eker rat model, but rapamycin is unlikely to eradicate all disease as a result of the development of drug resistance. Our data also suggest the role of a rapamycininsensitive pathway during tumor initiation. (Pediatr Res 57: 67-75, 2005) Abbreviations AML, angiomyolipoma mTOR, mammalian target of rapamycin TSC, tuberous sclerosis complex TUNEL, transferase-mediated dUTP nick-end labeling VEGF, vascular endothelial growth factor 4E-BP1, 4E-binding protein 1
The FHIT gene has been implicated as a tumor suppressor gene in human malignancies. To determine ... more The FHIT gene has been implicated as a tumor suppressor gene in human malignancies. To determine if FHIT alterations play a role in human squamous cell carcinogenesis of the head and neck (HNSCC), we examined the gene and its product by RT ± PCR, SSCP, Northern, Southern, and Western blot analysis in primary HNSCC and/or HNSCC cell lines. Three of 32 tumor samples lacked detectable expression of FHIT by RT ± PCR but showed ampli®cation of a control gene of similar size. One of 29 primary tumors and 2/9 HNSCC cell lines exhibited aberrant transcripts generated by RT ± PCR methods using one set of 40 cycles of ampli®cation. FHIT mRNA expression was absent in seven HNSCC cell lines but detectable in primary keratinocytes by Northern analysis. Using speci®c polyclonal antiserum to the full-length FHIT protein in immunoblot analyses, 4/9 cell lines analysed showed no expression of pFhit, two exhibited low levels of expression, and three expressed a putative truncated pFhit. One of 15 tumors analysed also exhibited an overexpressed truncated protein. PCR/SSCP and Southern analysis of one cell line DNA that expressed a truncated protein indicated that it sustained homozygous loss of FHIT exon 5. Our results suggest that alterations in FHIT at the DNA, RNA, and protein levels exist at a low but signi®cant frequency in HNSCCs. Further studies regarding the potential biological activity of FHIT are needed to clarify the role of this gene in HNSCC tumorigenesis.
Fibrolamellar hepatocellular carcinoma (FL-HCC) has historically been classified as a rare subtyp... more Fibrolamellar hepatocellular carcinoma (FL-HCC) has historically been classified as a rare subtype of HCC. However, unlike "classic" HCC, it occurs in children and young adults without underlying liver disease. The recent discovery of a deletion mutation in all FL-HCCs represented a major advancement in understanding the pathogenesis of this disease. This deletion results in the fusion of the genes encoding a heat shock protein (DNAJB1) and the catalytic subunit of protein kinase A (PKA, PRKACA), and overexpression of PRKACA and enhanced cAMP-dependent PKA activity. This review summarizes recent advancements in FL-HCC pathogenesis and characteristics of the HSP40-PKA C protein.
Angiomyolipomata (AML) belong to a family of tumors known as perivascular epithelioid cell tumors... more Angiomyolipomata (AML) belong to a family of tumors known as perivascular epithelioid cell tumors (PEComas) that share a common immunophenotypic profile of muscle and melanocytic differentiation. These tumors are clonal in nature and have a strong association with tuberous sclerosis. Genetic analyses have reported allelic imbalance at the TSC2 locus on 16p13. In the context of non-TSC, non-LAM associated AMLs and non-renal PEComas, the functional status of the TSC2 signaling pathway has not been reported. Studies over the last several years have uncovered a critical role of the TSC1/2 genes in negatively regulating the Rheb/mTOR/p70S6K cascade. Here, we examined the activity of this pathway in sporadic AMLs and PEComas using immunohistochemical and biochemical analyses. We found increased levels of phospho-p70S6K, a marker of mTOR activity, in 15 of 15 non-TSC AMLs. This was accompanied by reduced phospho-AKT expression, a pattern that is consistent with the disruption of TSC1/2 function. Western blot analysis confirmed mTOR activation concurrent with the loss of TSC2 and not TSC1 in sporadic AMLs. Similarly, elevated phospho-p70S6K and reduced phospho-AKT expression was detected in 14/15 cases of extra-renal PEComas. These observations provide the first functional evidence that mTOR activation is common to sporadic, non-TSC-related AMLs and PEComas. This suggests the possibility that mTOR inhibitors such as rapamycin may be therapeutic for this class of disease.
Journal of the National Cancer Institute, Mar 18, 1998
Background: The fragile histidine triad (FHIT) gene at chromosome 3p14.2 has been proposed to be ... more Background: The fragile histidine triad (FHIT) gene at chromosome 3p14.2 has been proposed to be a candidate tumor suppressor gene in human cancers. To test whether FHIT exhibits the functional properties of a tumor suppressor gene, we studied the expression of its protein (pFHIT) in human carcinoma cells and examined the ability of FHIT to inhibit the neoplastic phenotype of cancer cells. Methods: Subcellular localization and patterns of protein expression in tumor cells were determined by immunohistochemical analysis and immunoblotting with the use of polyclonal anti-pFHIT antisera. In tumor cells with undetectable pFHIT, we examined the effect of recombinant pFHIT expression on morphology, growth rate, colony formation, and in vivo tumor formation. Results: We demonstrated that pFHIT is a cytoplasmic 17-kd polypeptide whose expression could not be detected in 30 of 52 human carcinoma cell lines tested. We observed, however, that the stable overexpression of pFHIT did not alter cell morphology, inhibit colony formation, or inhibit cell proliferation in vitro. Furthermore, overexpression of pFHIT did not lead to altered cell cycle kinetics in dividing cells. The in vivo tumorigenicity of a tumor cell line that expressed high levels of recombinant pFHIT was equivalent to that of control transfectants and of parental cells. Conclusions: These results suggest that the replacement of pFHIT in human carcinoma cells does not suppress tumor cell growth and that this protein may be involved in tumorigenesis in ways that are distinct from the ''classic'' tumor suppressor paradigm. [
Despite advances in interventional procedures and chemotherapeutic drug development, hepatocellul... more Despite advances in interventional procedures and chemotherapeutic drug development, hepatocellular carcinoma (HCC) is still the fourth leading cause of cancer-related deaths worldwide with a <30% 5-year survival rate. This poor prognosis can be attributed to the fact that HCC most commonly occurs in patients with pre-existing liver conditions, rendering many treatment options too aggressive. Patient survival rates could be improved by a more targeted approach. Ultrasound-induced cavitation can provide a means for overcoming traditional barriers defining drug uptake. The goal of this work was to evaluate preclinical efficacy of image-guided, cavitation-enabled drug delivery with a clinical ultrasound scanner. To this end, ultrasound conditions (unique from those used in imaging) were designed and implemented on a Philips EPIQ and S5-1 phased array probe to produced focused ultrasound for cavitation treatment. Sonovue ® microbubbles which are clinically approved as an ultrasound contrast agent were used for both imaging and cavitation treatment. A genetically engineered mouse model was bred and used as a physiologically relevant preclinical analog to human HCC. It was observed that imageguided and targeted microbubble cavitation resulted in selective disruption of the tumor blood flow and enhanced doxorubicin uptake and penetration. Histology results indicate that no gross morphological damage occurred as a result of this process. The combination of these effects may be exploited to treat HCC and other challenging malignancies and could be implemented with currently available ultrasound scanners and reagents.
Background: The potential health effects of polybrominated diphenyl ethers (PBDEs) that are widel... more Background: The potential health effects of polybrominated diphenyl ethers (PBDEs) that are widely used as flame-retardants in consumer products have been attributed, in part, to their endocrine disrupting properties. The purpose of this study is to examine the in vivo effects of an early exposure to PBDEs on the development of insulin resistance in mice. Results: The metabolic consequences of BDE-47 in mice with varying insulin sensitivities secondary to liver-specific activation of Akt (Pten fl/fl ;Alb Cre) and mTORC1 (Tsc1 fl/fl ;Alb Cre) as well as wild-type littermates, were studied. BDE-47, a dominant congener of PBDE, was given daily (1 mg/kg/day) for six weeks by oral gavage in young mice following weaning. At the end of the exposure, there were no significant differences in total body, liver, or white adipose tissue weights between the BDE-47-treated vs. DMSO-treated mice for each respective genotype. Metabolic studies revealed significant impairment in insulin sensitivity in the BDE-47-treated Pten fl/fl ;Alb Cre mice, but not in wild-type or Tsc1 fl/fl ;Alb Cre mice. This was not accompanied by significant alterations in plasma insulin levels or hepatic triglyceride accumulation in the Pten fl/fl ;Alb Cre mice. The mean plasma BDE-47 level in the wild-type mice was 11.7 ± 2.9 ng/g (wet weight). Conclusions: Our findings indicate that BDE-47 exposure during the early post-natal period induces a mild disturbance in glucose metabolism in susceptible mice with increased baseline insulin sensitivity. These results suggest an interaction between BDE-47 and genetic factors that regulate insulin signaling, which may result in long-term consequences.
With the widespread adoption of molecular profiling in clinical oncology practice, many physician... more With the widespread adoption of molecular profiling in clinical oncology practice, many physicians are faced with making therapeutic decisions based upon isolated genomic alterations. For example, epidermal growth factor receptor tyrosine kinase inhibitors (TKIs) are effective in EGFR-mutant non-small cell lung cancers (NSCLC) while anti-EGFR monoclonal antibodies are ineffective in Rasmutant colorectal cancers. The matching of mutations with drugs aimed at their respective gene products represents the current state of "precision" oncology. Despite the great expectations of this approach, only a fraction of cancers responds to 'targeted' interventions, and many early responders will ultimately develop resistance to these agents. The underwhelming success of mutation-driven therapies across all cancer types is not due to an inability to detect genetic changes in tumors; rather a deficit in functional insight into the genomic alterations that give rise to each cancer. The Achilles heel of precision oncology thus remains the lack of a robust functional understanding of an individual cancer genome that then allows prediction of the best therapy and resultant outcome for that patient. Current practice focuses on one 'actionable' mutation at a time, while solid cancers typically possess many mutations that involve different cellular sub-populations within a tumor. No method or platform currently exists to guide the interpretation of these complex data, nor to accurately predict response to treatment. This problem is particularly germane to primary liver cancers (PLC), for which only a handful of targeted therapies have been introduced. Here, we will review strategies aimed at overcoming some of these challenges in precision oncology, using liver cancer as an example.
Insulin resistance is an important contributing factor in non-alcoholic fatty liver disease. AKT ... more Insulin resistance is an important contributing factor in non-alcoholic fatty liver disease. AKT and mTORC1 are key components of the insulin pathway, and play a role in promoting de novo lipogenesis. However, mTORC1 hyperactivity per se does not induce steatosis in mouse livers, but instead, protects against high-fat diet induced steatosis. Here, we investigate the in vivo mechanism of steatosis-resistance secondary to mTORC1 activation, with emphasis on the role of S6K1-mediated feedback inhibition of AKT. Mice with single or double deletion of Tsc1 and/or S6k1 in a liver-specific or whole-body manner were generated to study glucose and hepatic lipid metabolism between the ages of 6-14 weeks. Following 8 weeks of high-fat diet, the Tsc1-/-;S6k1-/-mice had lower body weights but higher liver TG levels compared to that of the Tsc1-/-mice. However, the loss of S6k1 did not relieve feedback inhibition of Akt activity in the Tsc1-/-livers. To overcome Akt suppression, Pten was deleted in Tsc1-/-livers, and the resultant mice showed improved glucose tolerance compared with the Tsc1-/-mice. However, liver TG levels were significantly reduced in the Tsc1-/-;Pten-/-mice compared to the Pten-/-mice, which was restored with rapamycin. We found no correlation between liver TG and serum NEFA levels. Expression of lipogenic genes (Srebp1c, Fasn) were elevated in the Tsc1-/-;Pten-/-livers, but this was counterbalanced by an up-regulation of Cpt1a involved in fatty acid oxidation and the anti-oxidant protein, Nrf2. In summary, our in vivo models showed that mTORC1-induced resistance to steatosis was dependent on S6K1 activity, but not secondary to AKT suppression. These findings confirm that AKT and mTORC1 have opposing effects on hepatic lipid metabolism in vivo.
Proceedings of the National Academy of Sciences of the United States of America, Dec 22, 1998
Tuberous sclerosis is an autosomal dominant disorder characterized by the development of aberrant... more Tuberous sclerosis is an autosomal dominant disorder characterized by the development of aberrant growths in many tissues and organs. Linkage analysis revealed two disease-determining genes on chromosome 9 and chromosome 16. The tuberous sclerosis complex gene-2 (TSC2) on chromosome 16 encodes the tumor suppressor protein tuberin. We have shown earlier that loss of TSC2 is sufficient to induce quiescent cells to enter the cell cycle. Here we show that TSC2-negative fibroblasts exhibit a shortened G 1 phase. Although the expression of cyclin E, cyclin A, p21, or Cdc25A is unaffected, TSC2-negative cells express much lower amounts of the cyclin-dependent kinase (CDK) inhibitor p27 because of decreased protein stability. In TSC2 mutant cells the amount of p27 bound to CDK2 is diminished, accompanied with elevated kinase activity. Ectopic expression studies revealed that the aforementioned effects can be reverted by transfecting TSC2 in TSC2-negative cells. High ectopic levels of p27 have cell cycle inhibitory effects in TSC2-positive cells but not in TSC2-negative counterparts, although the latter still depend on CDK2 activity. Loss of TSC2 induces soft agar growth of fibroblasts, a process that cannot be inhibited by high levels of p27. Both phenotypes of TSC2-negative cells, their resistance to the activity of ectopic p27, and the instability of endogenous p27, could be explained by our observation that the nucleoprotein p27 is mislocated into the cytoplasm upon loss of TSC2. These findings provide insights into the molecular mechanism of how loss of TSC2 induces cell cycle entry and allow a better understanding of its tumor suppressor function. MATERIALS AND METHODS Cells, Cell Culture, Flow Cytometry, and Centrifugal Elutriation. EEF4 (TSC2-positive) and EEF8 (TSC2-negative) cells were derived from Eker rat embryos homozygous for the wild-type and the Eker-mutant TSC2 gene, respectively. Whole embryos were removed on day 10.5 before in utero deaths of the Eker homozygous mutants have occurred. Sam-The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ''advertisement'' in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Organotypic tumor slices represent a physiologically-relevant culture system for studying the tum... more Organotypic tumor slices represent a physiologically-relevant culture system for studying the tumor microenvironment. Systematic characterization of the tumor slice culture system will enable its effective application for translational research. Here, using flow cytometry-based immunophenotyping, we performed a comprehensive characterization of the immune cell composition in organotypic tumor slices prepared from four syngeneic mouse tumor models and a human liver tumor. We found that the immune cell compositions of organotypic tumor slices prepared on the same day as the tumor cores were harvested are similar. Differences were primarily observed in the lymphocyte population of a clinical hepatocellular carcinoma case. Viable populations of immune cells persisted in the tumor slices for 7 days. Despite some changes in the immune cell populations, we showed the utility of mouse tumor slices for assessing responses to immune-modulatory agents. Further, we demonstrated the ability to use patient-derived xenograft tumor slices for assessing responses to targeted and cytotoxic drugs. Overall, tumor slices provide a broadly useful platform for studying the tumor microenvironment and evaluating the preclinical efficacy of cancer therapeutics.
Objective Programmed cell death protein 1 (PD-1) checkpoint inhibition and adoptive cellular ther... more Objective Programmed cell death protein 1 (PD-1) checkpoint inhibition and adoptive cellular therapy have had limited success in patients with microsatellite stable colorectal cancer liver metastases (CRLM). We sought to evaluate the effect of interleukin 10 (IL-10) blockade on endogenous T cell and chimeric antigen receptor T (CAR-T) cell antitumour function in CRLM slice cultures. Design We created organotypic slice cultures from human CRLM (n=38 patients' tumours) and tested the antitumour effects of a neutralising antibody against IL-10 (αIL-10) both alone as treatment and in combination with exogenously administered carcinoembryonic antigen (CEA)-specific CART cells. We evaluated slice cultures with single and multiplex immunohistochemistry, in situ hybridisation, single-cell RNA sequencing, reverse-phase protein arrays and time-lapse fluorescent microscopy. Results αIL-10 generated a 1.8-fold increase in T cell-mediated carcinoma cell death in human CRLM slice cultures. αIL-10 significantly increased proportions of CD8 + T cells without exhaustion transcription changes, and increased human leukocyte antigen-DR isotype (HLA-DR) expression of macrophages. The antitumour effects of αIL-10 were reversed by major histocompatibility complex class I or II (MHC-I or MHC-II) blockade, confirming the essential role of antigen presenting cells. Interrupting IL-10 signalling also rescued murine CART cell proliferation and cytotoxicity from myeloid cell-mediated immunosuppression. In human CRLM slices, αIL-10 increased CEA-specific CART cell activation and CART cell-mediated cytotoxicity, with nearly 70% carcinoma cell apoptosis across multiple human tumours. Pretreatment with an IL-10 receptor blocking antibody also potentiated CART function. Conclusion Neutralising the effects of IL-10 in human CRLM has therapeutic potential as a stand-alone treatment and to augment the function of adoptively transferred CART cells. HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY ⇒ IL-10 represents a major immunosuppressive signal to overcome in patients with CRLM. Targeting IL-10/IL-10 receptor signalling has great potential to be incorporated into systemic therapy for patients with immune checkpoint blockade-resistant, MSS CRLM. CART cells, armed against IL-10 suppression, may pave the way for more effective adoptive cell therapies for patients with MSS CRLM refractory to currently available surgical and medical therapies.
ABSTRACTThe DNAJ-PKAc fusion kinase is a defining feature of the adolescent liver cancer fibrolam... more ABSTRACTThe DNAJ-PKAc fusion kinase is a defining feature of the adolescent liver cancer fibrolamellar carcinoma (FLC). A single lesion on chromosome 19 generates this mutant kinase by creating a fused gene encoding the chaperonin binding domain of Hsp40 (DNAJ) in frame with the catalytic core of protein kinase A (PKAc). FLC tumors are notoriously resistant to standard chemotherapies. Aberrant kinase activity is assumed to be a contributing factor. Yet recruitment of binding partners, such as the chaperone Hsp70, implies that the scaffolding function of DNAJ- PKAc may also underlie pathogenesis. By combining proximity proteomics with biochemical analyses and photoactivation live-cell imaging we demonstrate that DNAJ-PKAc is not constrained by A-kinase anchoring proteins. Consequently, the fusion kinase phosphorylates a unique array of substrates. One validated DNAJ-PKAc target is the Bcl-2 associated athanogene 2 (BAG2), a co-chaperone recruited to the fusion kinase through associat...
Hepatocellular carcinoma (HCC) is a significant cause of morbidity and mortality worldwide with l... more Hepatocellular carcinoma (HCC) is a significant cause of morbidity and mortality worldwide with limited therapeutic options for advanced disease. Targeted alpha therapy (TAT) is an emerging class of targeted cancer therapy in which alpha-particle-emitting radionuclides, such as thorium-227, are specifically delivered to cancer tissue. Glypican-3 (GPC3) is a cell surface glycoprotein highly expressed on HCC. In this study, we describe the development and in vivo efficacy of a 227 Th-labeled GPC3 targeting antibody conjugate (227 Th-octapa-αGPC3) for treatment of HCC in an orthotopic murine model. METHODS The chelator p-SCN-Bn-H4octapa-NCS (octapa) was conjugated to a GPC3 targeting antibody (αGPC3) for subsequent 227 Th radiolabeling (octapa-αGPC3). Conditions were varied to optimize radiolabeling of 227 Th. In vitro stability was evaluated by measuring percentage of protein-bound 227 Th by gamma-ray spectroscopy. An orthotopic athymic Nu/J murine model using HepG2 cells was developed. Biodistribution and blood clearance of 227 Th-octapa-αGPC3 was evaluated in tumor bearing mice. Efficacy of 227 Th-octapa-αGPC3 was assessed in tumor bearing animals with serial measurement of serum alpha-fetoprotein at 23 days after radionuclide injection. RESULTS Octapa-conjugated αGPC3 provided up to 70% 227 Th labeling yield in 2 h at room temperature. In the presence of ascorbate, 97.8% of 227 Th was bound to αGPC3-octapa after 14 d in phosphate buffered saline. In HepG2 tumor-bearing mice, highly specific GPC3 targeting was observed, with significant 227 Th-octapa-αGPC3 accumulation in the tumor over time and minimal accumulation in normal tissue. 23 days after treatment, significant reduction in tumor burden was observed in 4 mice receiving 500 kBq/kg 227 Th-octapa-αGPC3 by tail vein injection. No acute off-target toxicity was observed and no animals died prior to termination of the study. CONCLUSION 227 Th-octapa-αGPC3 was observed to be stable in vitro, maintain high specificity for GPC3 with favorable biodistribution in vivo, and result in significant antitumor activity without significant acute off-target toxicity in an orthotopic murine model of HCC.
Glypican-3 (GPC3) is a tumor associated antigen expressed by hepatocellular carcinoma (HCC) cells... more Glypican-3 (GPC3) is a tumor associated antigen expressed by hepatocellular carcinoma (HCC) cells. This preclinical study evaluated the efficacy of a theranostic platform using a GPC3-targeting antibody αGPC3 conjugated to zirconium-89 (89Zr) and yttrium-90 (90Y) to identify, treat, and assess treatment response in a murine model of HCC. A murine orthotopic xenograft model of HCC was generated. Animals were injected with 89Zr-labeled αGPC3 and imaged with a small-animal positron emission/computerized tomography (PET/CT) imaging system (immuno-PET) before and 30 days after radioimmunotherapy (RIT) with 90Y-labeled αGPC3. Serum alpha fetoprotein (AFP), a marker of tumor burden, was measured. Gross tumor volume (GTV) and SUVmax by immuno-PET was measured using fixed intensity threshold and manual segmentation methods. Immuno-PET GTV measurements reliably quantified tumor burden prior to RIT, strongly correlating with serum AFP (R2 = 0.90). Serum AFP was significantly lower 30 days afte...
Direct-Acting Antiviral (DAA) drugs are highly effective in eliminating HCV in most chronically i... more Direct-Acting Antiviral (DAA) drugs are highly effective in eliminating HCV in most chronically infected subjects, but it is unclear whether the multiple mechanisms employed by HCV to disable both innate and adaptive immunity cease to function as soon as HCV is eliminated. To test this, we evaluated the capacity of human liver tissue to respond to TLR3 and TLR4 ligands using non-infected liver tissue, tissue with active HCV infection, and tissue from which HCV had been eliminated by DAA. We found that DAA-treated, formerly HCV-infected liver tissue manifested ongoing abnormalities of innate immunity that mapped to liver non-parenchymal cells (NPC). Hepatic innate immunity was not suppressed but enhanced in HCV-infected tissue, and these abnormalities were not corrected in the successfully DAA-treated liver tissue. In conclusion, ongoing immune activation persists in formerly HCV-infected but now DAA-cured liver.
With the widespread adoption of molecular profiling in clinical oncology practice, many physician... more With the widespread adoption of molecular profiling in clinical oncology practice, many physicians are faced with making therapeutic decisions based upon isolated genomic alterations. For example, epidermal growth factor receptor tyrosine kinase inhibitors (TKIs) are effective in EGFR-mutant non-small cell lung cancers (NSCLC) while anti-EGFR monoclonal antibodies are ineffective in Rasmutant colorectal cancers. The matching of mutations with drugs aimed at their respective gene products represents the current state of "precision" oncology. Despite the great expectations of this approach, only a fraction of cancers responds to 'targeted' interventions, and many early responders will ultimately develop resistance to these agents. The underwhelming success of mutation-driven therapies across all cancer types is not due to an inability to detect genetic changes in tumors; rather a deficit in functional insight into the genomic alterations that give rise to each cancer. The Achilles heel of precision oncology thus remains the lack of a robust functional understanding of an individual cancer genome that then allows prediction of the best therapy and resultant outcome for that patient. Current practice focuses on one 'actionable' mutation at a time, while solid cancers typically possess many mutations that involve different cellular sub-populations within a tumor. No method or platform currently exists to guide the interpretation of these complex data, nor to accurately predict response to treatment. This problem is particularly germane to primary liver cancers (PLC), for which only a handful of targeted therapies have been introduced. Here, we will review strategies aimed at overcoming some of these challenges in precision oncology, using liver cancer as an example.
Organotypic tumor slices represent a physiologically-relevant culture system for studying the tum... more Organotypic tumor slices represent a physiologically-relevant culture system for studying the tumor microenvironment. Systematic characterization of the tumor slice culture system will enable its effective application for translational research. Here, using flow cytometry-based immunophenotyping, we performed a comprehensive characterization of the immune cell composition in organotypic tumor slices prepared from four syngeneic mouse tumor models and a human liver tumor. We found that the immune cell compositions of organotypic tumor slices prepared on the same day as the tumor cores were harvested are similar. Differences were primarily observed in the lymphocyte population of a clinical hepatocellular carcinoma case. Viable populations of immune cells persisted in the tumor slices for 7 days. Despite some changes in the immune cell populations, we showed the utility of mouse tumor slices for assessing responses to immune-modulatory agents. Further, we demonstrated the ability to use patient-derived xenograft tumor slices for assessing responses to targeted and cytotoxic drugs. Overall, tumor slices provide a broadly useful platform for studying the tumor microenvironment and evaluating the preclinical efficacy of cancer therapeutics.
Fibrolamellar carcinoma (FLC) is a rare liver cancer. FLCs uniquely produce DNAJ-PKAc, a chimeric... more Fibrolamellar carcinoma (FLC) is a rare liver cancer. FLCs uniquely produce DNAJ-PKAc, a chimeric enzyme consisting of a chaperonin-binding domain fused to the Cα subunit of protein kinase A. Biochemical analyses of clinical samples reveal that a unique property of this fusion enzyme is the ability to recruit heat shock protein 70 (Hsp70). This cellular chaperonin is frequently up-regulated in cancers. Gene-editing of mouse hepatocytes generated disease-relevant AML12DNAJ-PKAc cell lines. Further analyses indicate that the proto-oncogene A-kinase anchoring protein-Lbc is up-regulated in FLC and functions to cluster DNAJ-PKAc/Hsp70 sub-complexes with a RAF-MEK-ERK kinase module. Drug screening reveals Hsp70 and MEK inhibitor combinations that selectively block proliferation of AML12DNAJ-PKAc cells. Phosphoproteomic profiling demonstrates that DNAJ-PKAc biases the signaling landscape toward ERK activation and engages downstream kinase cascades. Thus, the oncogenic action of DNAJ-PKAc ...
Cellular and Molecular Gastroenterology and Hepatology, 2019
Small RNA profiling of primary fibrolamellar carcinoma tumors shows microRNA-375 (miR-375) as the... more Small RNA profiling of primary fibrolamellar carcinoma tumors shows microRNA-375 (miR-375) as the most dysregulated miRNA. Several disease models recapitulate the dramatic suppression of miR-375. Functional interrogation of miR-375 in a novel fibrolamellar carcinoma cell line shows that it suppresses fibrolamellar carcinoma cell growth and migration. BACKGROUND & AIMS: Fibrolamellar carcinoma (FLC) is a rare liver cancer that primarily affects adolescents and young adults. It is characterized by a heterozygous approximately 400-kb deletion on chromosome 19 that results in a unique fusion between DnaJ heat shock protein family member B1 (DNAJB1) and the alpha catalytic subunit of protein kinase A (PRKACA). The role of microRNAs (miRNAs) in FLC remains unclear. We identified dysregulated miRNAs in FLC and investigated whether dysregulation of 1 key miRNA contributes to FLC pathogenesis. METHODS: We analyzed small RNA sequencing (smRNA-seq) data from The Cancer Genome Atlas to identify dysregulated miRNAs in primary FLC tumors and validated the findings in 3 independent FLC cohorts. smRNA-seq also was performed on a FLC patient-derived xenograft model as well as purified cell populations of the liver to determine whether key miRNA changes were tumor cell-intrinsic. We then used clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (Cas9) technology and transposon-mediated gene transfer in mice to determine if the presence of DNAJB1-PRKACA is sufficient to suppress miR-375 expression. Finally, we established a new FLC cell line and performed colony formation and scratch wound assays to determine the functional consequences of miR-375 overexpression. RESULTS: We identified miR-375 as the most dysregulated miRNA in primary FLC tumors (27-fold down-regulation; P ¼ .009). miR-375 expression also was decreased significantly in a FLC patient-derived xenograft model compared to 4 different cell populations of the liver. Introduction of DNAJB1-PRKACA by clustered regularly interspaced short palindromic repeats/CRISPRassociated protein 9 engineering and transposon-mediated somatic gene transfer in mice was sufficient to induce significant loss of miR-375 expression (P < .05). Overexpression of miR-375 in FLC cells inhibited Hippo signaling pathway proteins, including yes-associated protein 1 and connective tissue growth factor, and suppressed cell proliferation and migration (P < .05). CONCLUSIONS: We identified miR-375 as the most downregulated miRNA in FLC tumors and showed that overexpression of miR-375 mitigated tumor cell growth and invasive potential. These findings open a potentially new molecular therapeutic approach. Further studies are necessary to determine how DNAJB1-PRKACA suppresses miR-375 expression and whether miR-375 has additional important targets in this tumor.
Tuberous sclerosis complex (TSC) presents in the pediatric population with a constellation of ben... more Tuberous sclerosis complex (TSC) presents in the pediatric population with a constellation of benign tumors that affect the brain, heart, kidney, lung, and skin. No therapy has been shown to halt disease progression or to prevent its onset. The pathogenesis of TSC stems from the inactivation of one of the two TSC genes, TSC1 and TSC2. A key function of these genes is to regulate the mammalian target of rapamycin (mTOR) pathway in response to cellular energy and nutrient and growth factor availability. Consequently, TSC-related tumors exhibit uncontrolled activation of mTOR and its effectors. Previous work has shown that a specific mTOR inhibitor, rapamycin, effectively downregulated mTOR activity in renal tumors of Eker rats that carry a germline Tsc2 mutation. Using this model, we investigated the effects of rapamycin on pituitary and renal tumors. We observed that rats with pituitary tumors had significantly shorter survival than those without pituitary pathology. Treatment with rapamycin effectively improved their clinical state and prolonged their survival. Rapamycin also resulted in a significant decrease in the size of the Tsc2-related renal tumors. In both types of pathology, tumor response was accompanied by down-regulation of ribo-somal S6 kinase activity, reduction in cell size, and induction of apoptosis. Evidence for drug resistance was found in a small percentage of lesions after prolonged therapy. When rapamycin was given before onset of disease, subsequent development of macroscopic renal tumors was reduced, but no effect on the number of microscopic precursor lesions was found. We conclude that rapamycin-sensitive mTOR activity was critical to tumor progression in the Eker rat model, but rapamycin is unlikely to eradicate all disease as a result of the development of drug resistance. Our data also suggest the role of a rapamycininsensitive pathway during tumor initiation. (Pediatr Res 57: 67-75, 2005) Abbreviations AML, angiomyolipoma mTOR, mammalian target of rapamycin TSC, tuberous sclerosis complex TUNEL, transferase-mediated dUTP nick-end labeling VEGF, vascular endothelial growth factor 4E-BP1, 4E-binding protein 1
The FHIT gene has been implicated as a tumor suppressor gene in human malignancies. To determine ... more The FHIT gene has been implicated as a tumor suppressor gene in human malignancies. To determine if FHIT alterations play a role in human squamous cell carcinogenesis of the head and neck (HNSCC), we examined the gene and its product by RT ± PCR, SSCP, Northern, Southern, and Western blot analysis in primary HNSCC and/or HNSCC cell lines. Three of 32 tumor samples lacked detectable expression of FHIT by RT ± PCR but showed ampli®cation of a control gene of similar size. One of 29 primary tumors and 2/9 HNSCC cell lines exhibited aberrant transcripts generated by RT ± PCR methods using one set of 40 cycles of ampli®cation. FHIT mRNA expression was absent in seven HNSCC cell lines but detectable in primary keratinocytes by Northern analysis. Using speci®c polyclonal antiserum to the full-length FHIT protein in immunoblot analyses, 4/9 cell lines analysed showed no expression of pFhit, two exhibited low levels of expression, and three expressed a putative truncated pFhit. One of 15 tumors analysed also exhibited an overexpressed truncated protein. PCR/SSCP and Southern analysis of one cell line DNA that expressed a truncated protein indicated that it sustained homozygous loss of FHIT exon 5. Our results suggest that alterations in FHIT at the DNA, RNA, and protein levels exist at a low but signi®cant frequency in HNSCCs. Further studies regarding the potential biological activity of FHIT are needed to clarify the role of this gene in HNSCC tumorigenesis.
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Papers by Raymond Yeung