Chromosome 8p engineering reveals increased metastatic potential targetable by patient-specific synthetic lethality in liver cancer.

Large-scale chromosomal aberrations are prevalent in human cancer, but their function remains poorly understood. We established chromosome-engineered hepatocellular carcinoma cell lines using CRISPR-Cas9 genome editing. A 33-mega-base pair region on chromosome 8p (chr8p) was heterozygously deleted, mimicking a frequently observed chromosomal deletion. Using this isogenic model system, we delineated the functional consequences of chr8p loss and its impact on metastatic behavior and patient survival. We found that metastasis-associated genes on chr8p act in concert to induce an aggressive and invasive phenotype characteristic for chr8p-deleted tumors. Genome-wide CRISPR-Cas9 viability screening in isogenic chr8p-deleted cells served as a powerful tool to find previously unidentified synthetic lethal targets and vulnerabilities accompanying patient-specific chromosomal alterations. Using this target identification strategy, we showed that chr8p deletion sensitizes tumor cells to targeting of the reactive oxygen sanitizing enzyme Nudix hydrolase 17. Thus, chromosomal engineering allowed for the identification of novel synthetic lethalities specific to chr8p loss of heterozygosity.

Karyopherin α2-dependent import of E2F1 and TFDP1 maintains protumorigenic stathmin expression in liver cancer.

BACKGROUND: Members of the karyopherin superfamily serve as nuclear transport receptors/adaptor proteins and provide exchange of macromolecules between the nucleo- and cytoplasm. Emerging evidence suggests a subset of karyopherins to be dysregulated in hepatocarcinogenesis including karyopherin-α2 (KPNA2). However, the functional and regulatory role of KPNA2 in liver cancer remains incompletely understood. METHODS: Quantitative proteomics (LC-MS/MS, ~ 1750 proteins in total) was used to study changes in global protein abundance upon siRNA-mediated KPNA2 knockdown in HCC cells. Functional and mechanistic analyses included colony formation and 2D migration assays, co-immunoprecipitation (CoIP), chromatin immunoprecipitation (ChIP), qRT-PCR, immmunblotting, and subcellular fractionation. In vitro results were correlated with data derived from a murine HCC model and HCC patient samples (3 cohorts, n > 600 in total). RESULTS: The proteomic approach revealed the pro-tumorigenic, microtubule (MT) interacting protein stathmin (STMN1) among the most downregulated proteins upon KPNA2 depletion in HCC cells. We further observed that KPNA2 knockdown leads to reduced tumor cell migration and colony formation of HCC cells, which could be phenocopied by direct knockdown of stathmin. As the underlying regulatory mechanism, we uncovered E2F1 and TFDP1 as transport substrates of KPNA2 being retained in the cytoplasm upon KPNA2 ablation, thereby resulting in reduced STMN1 expression. Finally, murine and human HCC data indicate significant correlations of STMN1 expression with E2F1/TFPD1 and with KPNA2 expression and their association with poor prognosis in HCC patients. CONCLUSION: Our data suggest that KPNA2 regulates STMN1 by import of E2F1/TFDP1 and thereby provide a novel link between nuclear transport and MT-interacting proteins in HCC with functional and prognostic significance.

Perilipin 1 Expression Differentiates Liposarcoma from Other Types of Soft Tissue Sarcoma.

Lipid droplets, a morphologic feature of adipocytic tumors, are strongly regulated by associated proteins of the perilipin/PAT (perilipin, adipophilin, and tail-interacting protein of 47 kD) family. So far, the use of perilipins as markers for differential diagnosis of soft tissue tumors has only been studied in a few cases. The aim of this study was to investigate the expression of perilipins in 478 human soft tissue tumors and 60 respective normal tissues. Perilipin 1 was immunohistochemically positive in all studied cases of well-differentiated liposarcomas, >90% of myxoid round cell liposarcomas, and >70% of pleomorphic liposarcomas, whereas only the differentiated components of dedifferentiated liposarcomas were immunohistochemically positive for perilipin 1. All other types of soft tissue sarcomas were negative for perilipin 1. Perilipin 2 was more prominent in dedifferentiated and pleomorphic liposarcomas and nearly all other high-grade sarcomas. In well-differentiated liposarcomas, lipomas, or normal adipose tissue, perilipin 2 was virtually absent. In addition, long-term stimulation of adipogenesis in the liposarcoma cell line LiSa-2 restored perilipin 1 expression, as exhibited in the source tumor. Furthermore, knockdown of perilipin 2 or perilipin 3 in LiSa-2 cells influenced lipid droplet number and size as well as cell vitality. In summary, perilipin 1 is a promising marker for the differential diagnosis of liposarcomas from other soft tissue sarcomas, whereas perilipin 2 correlates negatively with tumor grade and may be therapeutically useful.

Proteomic Analysis Reveals GMP Synthetase as p53 Repression Target in Liver Cancer.

Disruption of the tumor-suppressive p53 network is a key event in human malignancies, including primary liver cancer. In response to different types of stress, p53 mediates several antiproliferative cellular outcomes, such as cell cycle arrest, apoptosis, and senescence, by activation or repression of its target genes. Metabolic alterations initiating or being part of the p53 response have become an actively studied research area in the p53 field, with several aspects that still remain to be elucidated. Herein, we identified GMP synthetase (GMPS), a key enzyme of de novo purine biosynthesis, as an important p53 repression target using a large-scale proteomics approach. This p53-mediated repression of GMPS could be validated by immunoblotting in Sk-Hep1, HepG2, and HuH6 cells. Moreover, we found GMPS transcriptionally repressed in a p21-dependent manner and its repression maintained in the context of p53-mediated cellular senescence. More important, direct knockdown of GMPS by RNA interference resulted in reduced cell viability and was sufficient to trigger cellular senescence. Finally, by comparing murine hepatocellular carcinomas, which developed in p53 wild-type (+/+) versus p53 null (-/-) mice, we observed higher GMPS expression in the latter, supporting the in vivo relevance of our findings. We conclude that repression of GMPS by p53 through p21 is a functionally relevant part of the p53-mediated senescence program limiting tumor cell growth in liver cancer.

Cellular apoptosis susceptibility (CAS) is linked to integrin ß1 and required for tumor cell migration and invasion in hepatocellular carcinoma (HCC).

Importins and exportins represent an integral part of the nucleocytoplasmic transport machinery with fundamental importance for eukaryotic cell function. A variety of malignancies including hepatocellular carcinoma (HCC) show de-regulation of nuclear transport factors such as overexpression of the exportin Cellular Apoptosis Susceptibility (CAS). The functional implications of CAS in hepatocarcinogenesis remain, however, poorly understood. Here we integrated proteomics, transcriptomics and functional assays with patient data to further characterize the role of CAS in HCC. By analyzing ~ 1700 proteins using quantitative mass spectrometry in HCC cells we found that CAS depletion by RNAi leads to de-regulation of integrins, particularly down-regulation of integrin ß1. Consistent with this finding, CAS knockdown resulted in substantially reduced migration and invasion of HCC cell lines as analyzed by 2D 'scratch' and invasion chamber assays, respectively. Supporting the potential in vivo relevance, high expression levels of CAS in HCC tissue samples were associated with macroangioinvasion and poorer patient outcome. Our data suggest a previously unanticipated link between CAS and integrin signaling which correlates with an aggressive HCC phenotype.

Cellular apoptosis susceptibility (CAS) is overexpressed in thyroid carcinoma and maintains tumor cell growth: A potential link to the BRAFV600E mutation.

Thyroid carcinoma is among the most common malignant endocrine neoplasms with a rising incidence. Genetic alterations occurring in thyroid cancer frequently affect the RAS/RAF/MEK/ERK-pathway such as the oncogenic, kinase-activating BRAF(V600E) mutation. Nuclear transport receptors including importins and exportins represent an important part of the nuclear transport machinery providing nucleo-cytoplasmic exchange of macromolecules. The role of nuclear transport receptors in the development and progression of thyroid carcinomas is largely unknown. Here, we studied the expression and function of the exportin cellular apoptosis susceptibility (CAS) in thyroid carcinogenesis and its link to the BRAF(V600E) mutation. By using immunohistochemistry (IHC) we found significantly increased IHC scores of CAS in primary papillary (PTC) and medullary (MTC), but not in follicular (FTC) thyroid carcinoma compared to non-tumorous (NT) thyroid tissue. Interestingly, metastases of the aforementioned subtypes including FTC showed a strong CAS positivity. Among PTCs we observed that CAS immunoreactivity was significantly higher in the tumors harboring the BRAF(V600E) mutation. Furthermore, depletion of CAS by RNAi in the BRAF(V600E)-positive PTC cell line B-CPAP led to reduced tumor cell growth measured by crystal violet assays. This phenotype could be attributed to reduced proliferation and increased cell death as assayed by BrdU ELISAs and immunoblotting for PARP-cleavage, respectively. Finally, we found additive effects of CAS siRNA and vemurafenib treatment in B-CPAP cells. Collectively, these data suggest that CAS overexpression in thyroid carcinoma depends on the subtype and the disease stage. Our findings also indicate that CAS maintains PTC cell proliferation and survival. Targeting CAS could represent a potential therapeutic approach particularly in combination with BRAF inhibitors such as vemurafenib in BRAF(V600E)-positive tumors.

Prosurvival function of the cellular apoptosis susceptibility/importin-α1 transport cycle is repressed by p53 in liver cancer.

UNLABELLED: Proteins of the karyopherin superfamily including importins and exportins represent an essential part of the nucleocytoplasmic transport machinery. However, the functional relevance and regulation of karyopherins in hepatocellular carcinoma (HCC) is poorly understood. Here we identified cellular apoptosis susceptibility (CAS, exportin-2) and its transport substrate importin-α1 (imp-α1) among significantly up-regulated transport factor genes in HCC. Disruption of the CAS/imp-α1 transport cycle by RNAi in HCC cell lines resulted in decreased tumor cell growth and increased apoptosis. The apoptotic phenotype upon CAS depletion could be recapitulated by direct knockdown of the X-linked inhibitor of apoptosis (XIAP) and partially reverted by XIAP overexpression. In addition, XIAP and CAS mRNA expression levels were correlated in HCC patient samples (r=0.463; P<0.01), supporting the in vivo relevance of our findings. Furthermore, quantitative mass spectrometry analyses of murine HCC samples (p53-/- versus p53+/+) indicated higher protein expression of CAS and imp-α1 in p53-/- tumors. Consistent with a role of p53 in regulating the CAS/imp-α1 transport cycle, we observed that both transport factors were repressed upon p53 induction in a p21-dependent manner. CONCLUSION: The CAS/imp-α1 transport cycle is linked to XIAP and is required to maintain tumor cell survival in HCC. Moreover, CAS and imp-α1 are targets of p53-mediated repression, which represents a novel aspect of p53's ability to control tumor cell growth in hepatocarcinogenesis.

Perilipin discerns chronic from acute hepatocellular steatosis.

BACKGROUND & AIMS: Hepatocellular steatosis is the most frequent liver disease in the western world and may develop further to steatohepatitis, liver cirrhosis and hepatocellular carcinoma. We have previously shown that lipid droplet (LD)-associated proteins of the perilipin/PAT-family are differentially expressed in hepatocyte steatosis and that perilipin is expressed de novo. The aim of this study was to determine the conditions for the temporal regulation of de novo synthesis of perilipin in vitro and in vivo. METHODS: Immunohistochemical PAT-analysis was performed with over 120 liver biopsies of different etiology and duration of steatosis. Steatosis was induced in cultured hepatocytic cells with combinations of lipids, steatogenic substances and DMSO for up to 40 days under conditions of stable down-regulation of adipophilin and/or TIP47. RESULTS: Whereas perilipin and adipophilin were expressed in human chronic liver disease irrespective of the underlying etiology, in acute/microvesicular steatosis TIP47, and MLDP were recruited from the cytoplasm to LDs, adipophilin was strongly increased, but perilipin was virtually absent. In long-term steatosis models in vitro, TIP47, MLDP, adipophilin, and finally perilipin were gradually induced. Perilipin and associated formation of LDs were intricately regulated on the transcriptional (PPARs, C/EBPs, SREBP), post-transcriptional, and post-translational level (TAG-amount, LD-fusion, phosphorylation-dependent lipolysis). In long-term steatosis models under stable down-regulation of adipophilin and/or TIP47, MLDP substituted for TIP47, and perilipin for adipophilin. CONCLUSIONS: LD-maturation in hepatocytes in vivo and in vitro involves sequential expression of TIP47, MLDP, adipophilin and finally perilipin. Thus, perilipin might be used for the differential diagnosis of chronic vs. acute steatosis.

Differential expression of E-prostanoid receptors in human hepatocellular carcinoma.

Recent studies have shown that inhibition of cyclooxygenases (e.g. COX-2) exerts antitumorigenic effects on hepatocellular carcinomas (HCCs), which are to a significant extent due to the abrogation of PGE(2) synthesis. PGE(2) acts via differentially regulated prostaglandin receptors (EP(1-4)). Our study was designed to investigate the expression pattern of EP-receptors in HCCs and to evaluate the therapeutic potential of selective EP-receptor antagonists. Using tissue microarrays including a total of 14 control livers, 17 liver cirrhoses, 22 premalignant dysplastic nodules (DNs) and 162 HCCs with different histological grades, the expression of COX-2, mPGES-1 and -2 and EP(1-4)-receptors was analyzed. Western immunoblot analyses were performed to confirm the expression in HCC cell lines. The effects of EP(1-4)-receptor antagonism on cell viability and apoptosis were investigated using MTT-assays and FACS-analyses, respectively. COX-2, mPGES-1 and -2 and EP(1-4)-receptors were expressed in all HCC tissues. COX-2 expression was highest in DNs and declined with loss of HCC-differentiation. With respect to COX-2 expression, a converse expression of EP(1-3) -receptors and mPGES-1 and -2 was found in DNs compared to HCCs. Selectively antagonizing EP(1)- and EP(3)-receptors reduced the viability of HCC cells in a dose-dependent manner, which was associated with apoptosis induction. Our results suggest a differential regulation of EP-receptor subtype expression with dedifferentiation of HCCs in which a converse expression pattern for COX-2 in comparison to EP(1-3)-receptors occurs. Of clinical interest, selectively antagonizing EP(1)- and EP(3)-receptors may provide a novel systemic therapeutic approach to the treatment of HCCs.

Ex vivo analysis of antineoplastic agents in precision-cut tissue slices of human origin: effects of cyclooxygenase-2 inhibition in hepatocellular carcinoma.

Cultures of precision-cut tissue slices allow the investigation of substance effects on human tissues under in vivo-like conditions over a limited time span. We have adapted the model for direct analyses of antineoplastic substances on tumor tissues. We have recently demonstrated that selective cyclooxygenase-2 (COX-2) inhibitors strongly suppress growth of human hepatocellular carcinoma (HCC) cells in vitro and nude mouse HCC implants by inducing apoptosis and reducing proliferation. We have now analyzed the effects of COX-2 inhibition on human tumor tissue. Three hundred micrometer slices of tumorous and non-tumorous liver tissue from three surgically resected HCCs were cultured with increasing concentrations of the selective COX-2 inhibitor Meloxicam (20-200 microM) for 6, 12, 24, and 48 h. The cultured tissue slices were analysed morphologically and by immunohistology for proliferation (Ki-67), apoptosis (M30), and COX-2 expression. COX-2 was expressed in all HCCs and in the non-tumorous liver tissue. Cytoplasmic COX-2 immunoreactivity in HCCs increased during culturing time. In two of three cases, COX-2 inhibition significantly increased tumor cell apoptosis in HCCs, whereas the low basal apoptosis rate in the non-tumorous liver parenchyma did not change. Tumor cell proliferation was mildly reduced, but the changes did not reach statistical significance. These results demonstrate that the precision-cut tissue slice culture model is a useful tool to analyze directly drug-dependent antitumorous or unwanted organ-specific effects. The analysis of COX-2 inhibition lends further support to the antineoplastic effects previously demonstrated in vitro and in animal models.