A hepatoprotective role of peritumoral non-parenchymal cells in early liver tumorigenesis.

Various 3D models of hepatocytes (HCs) have been established to assess liver functions in vitro. The contribution of the hepatic non-parenchymal cells (NPCs), however, is largely neglected in these models. Here, we report a comparative study of hepatic spheroids generated from freshly isolated mouse whole liver cells (WLCs) and HCs (referred to as SphWLC and SphHC, respectively). We found that HC differentiation was preserved better in SphWLC than in SphHC, and, when co-cultured with liver tumor spheroids (SphT), SphWLC showed more potent suppression of SphT growth compared to SphHC. Histological characterization revealed marked activation and accumulation of hepatic stellate cells (HSCs) at the SphWLC:SphT interface. We found that mixing HSCs in both 3D and 2D HC:tumor co-cultures provided potent protection to HCs against tumor-induced cell death. Activation of HSCs at the tumor border was similarly found in liver tumors from both mice and patients. Overall, our study suggests a hepatoprotective role of peritumoral HSCs in liver tumorigenesis and the potential application of SphWLC as a useful 3D model for dissecting the liver's response to tumorigenesis in vitro.

Targeting Aurora A Kinase (AAK) in Platinum-Resistant High Grade Serous Ovarian Cancer.

Aurora A kinase (AAK) involved in G2-M transition is functionally involved in centrosome maturation and maintaining an active spindle assembly checkpoint. We tested the hypothesis that in platinum-taxane resistant high grade serous ovarian cancer (HGSOC) inhibition of AAK involved in G2-M transition would enhance the anti-tumor activity of cisplatin (CP) or paclitaxel (PT). Using HGSOC cell lines from platinum-taxane refractory patients that do not harbor BRCA1/2 mutations, we tested the anti-tumor activity of CP, or PT alone or in combination with the AAK inhibitor alisertib (AL). Treatment with CP for 3 h or PT for 6 h followed sequentially by AL for 48 h led to a significant decrease in cell survival (p < 0.001) compared to treatment with either drug alone in HGSOC cells but not in immortalized normal human ovarian surface epithelium or normal human fallopian tube secretory epithelium cells. The treatment with CP or PT followed by AL also led to a significant increase in reactive oxygen species (p < 0.05), apoptosis (p < 0.001) and accumulation of cells in G2/M that was accompanied by a modest increase in expression of AAK. Downregulation of AAK, but not aurora B kinase, with targeted siRNAs also significantly enhanced apoptosis by CP or PT, suggesting that AL specifically targeted AAK. In summary, in HGSOC without BRCA1/2 mutations, CP, or PT resistance can potentially be circumvented by sequential treatment with AL that inhibits AAK involved in G2-M transition.

Increased expression of neurotensin in high grade serous ovarian carcinoma with evidence of serous tubal intraepithelial carcinoma.

High grade serous ovarian carcinoma (HGSC) without identifiable serous tubal intraepithelial carcinoma (STIC) within the fallopian tube (FT) occurs in approximately 50% of patients. The objective of this study was to use a multisite tumor sampling approach to study HGSC with and without STIC. RNAseq analysis of HGSC samples collected from multiple sites e.g. ovary, FT and peritoneum, revealed moderate levels of intrapatient heterogeneity in gene expression that could influence molecular profiles. Mixed-model ANOVA analysis of gene expression in tumor samples from patients with multiple tumor sites (n = 13) and patients with a single site tumor sample (n = 11) to compare HGSC-STIC to HGSC-NOSTIC identified neurotensin (NTS) as significantly higher (> two-fold change, False Discovery Rate (FDR) < 0.10) in HGSC-STIC. This data was validated using publicly available RNA-Seq datasets. Concordance between higher NTS gene expression and NTS peptide levels in HGSC-STIC samples was demonstrated by immunohistochemistry. To determine the role of NTS in HGSC, five ovarian cancer (OvCa) cell lines were screened for expression of NTS and its receptors, NTSR1 and NTSR3. Increased expression of NTS and NSTR1 was observed in several of the OvCa cells, whereas the NTSR3 receptor was lower in all OvCa cells, compared to immortalized FT epithelial cells. Treatment with NTSR1 inhibitor (SR48692) decreased cell proliferation, but increased cell migration in OvCa cells. The effects of SR48692 were receptor mediated, since transient RNAi knockdown of NTSR1 mimicked the migratory effects and knockdown of NTSR3 mimicked the anti-proliferative effects. Further, knockdown of NTSR1 or NTSR3 was associated with acquisition of distinct morphological phenotypes, epithelial or mesenchymal, respectively. Taken together, our results reveal a difference in a biologically active pathway between HGSC with and without STIC. Furthermore, we identify neurotensin signaling as an important pathway involved in cell proliferation and epithelial-mesenchymal transition in HGSC-STIC which warrants further study as a potential therapeutic target. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Clonal lineage of high grade serous ovarian cancer in a patient with neurofibromatosis type 1.

Neurofibromatosis type 1 (NF1) is caused by mutations in the NF1 gene encoding neurofibromin, which negatively regulates Ras signaling. NF1 patients have an increased risk of developing early onset breast cancer, however, the association between NF1 and high grade serous ovarian cancer (HGSOC) is unclear. Since most NF1-related tumors exhibit early biallelic inactivation of NF1, we evaluated the evolution of genetic alterations in HGSOC in an NF1 patient. Somatic variation analysis of whole exome sequencing of tumor samples from both ovaries and a peritoneal metastasis showed a clonal lineage originating from an ancestral clone within the left adnexa, which exhibited copy number (CN) loss of heterozygosity (LOH) in the region of chromosome 17 containing TP53, NF1, and BRCA1 and mutation of the other TP53 allele. This event led to biallelic inactivation of NF1 and TP53 and LOH for the BRCA1 germline mutation. Subsequent CN alterations were found in the dominant tumor clone in the left ovary and nearly 100% of tumor at other sites. Neurofibromin modeling studies suggested that the germline NF1 mutation could potentially alter protein function. These results demonstrate early, biallelic inactivation of neurofibromin in HGSOC and highlight the potential of targeting RAS signaling in NF1 patients.

HOXA4/HOXB3 gene expression signature as a biomarker of recurrence in patients with high-grade serous ovarian cancer following primary cytoreductive surgery and first-line adjuvant chemotherapy.

OBJECTIVES: Aberrant homeobox (HOX) gene expression is reported in high-grade serous ovarian carcinoma (HGSOC), however, its prognostic significance remains unclear. METHODS: HOX genes associated with progression-free survival (PFS) in a discovery cohort of primary HGSOC samples with RNA sequencing data, and those previously reported to be associated with clinical outcomes, were selected for qPCR testing in an independent training cohort of primary HGSOC samples (n=71). A prognostic model for PFS was developed using univariate and multivariate Cox regression. Patients were stratified into risk groups that optimized the test statistic. The model was tested in an independent HGSOC cohort from The Cancer Genome Atlas (TCGA) (n=320). The effect of selected HOX genes on drug sensitivity and reactive oxygen species (ROS) accumulation was examined in vitro. RESULTS: Of 23 HOX genes tested in the training cohort, HOXA4 (HR=1.20, 95% CI=1.07-1.34, P=0.002) and HOXB3 (HR=1.09, 95% CI=1.01-1.17, P=0.027) overexpression were significantly associated with shorter PFS in multivariate analysis. Based on the optimal cutoff of the HOXA4/HOXB3 risk score, median PFS was 16.9months (95% CI=14.6-21.2months) and not reached (>80months) for patients with high and low risk scores, respectively (HR=8.89, 95% CI=2.09-37.74, P<0.001). In TCGA, the HOXA4/HOXB3 risk score was significantly associated with disease-free survival (HR=1.44, 95% CI=1.00-2.09, P=0.048). HOXA4 or HOXB3 overexpression in ovarian cancer cells decreased sensitivity to cisplatin and attenuated the generation of cisplatin-induced ROS (P<0.05). CONCLUSIONS: HOXA4/HOXB3 gene expression-based risk score may be useful for prognostic risk stratification and warrants prospective validation in HGSOC patients.

Roles of the C-terminal domains of topoisomerase IIα and topoisomerase IIß in regulation of the decatenation checkpoint.

Topoisomerase (topo) IIα and IIß maintain genome stability and are targets for anti-tumor drugs. In this study, we demonstrate that the decatenation checkpoint is regulated, not only by topo IIα, as previously reported, but also by topo IIß. The decatenation checkpoint is most efficient when both isoforms are present. Regulation of this checkpoint and sensitivity to topo II-targeted drugs is influenced by the C-terminal domain (CTD) of the topo II isoforms and by a conserved non-catalytic tyrosine, Y640 in topo IIα and Y656 in topo IIß. Deletion of most of the CTD of topo IIα, while preserving the nuclear localization signal (NLS), enhances the decatenation checkpoint and sensitivity to topo II-targeted drugs. In contrast, deletion of most of the CTD of topo IIß, while preserving the NLS, and mutation of Y640 in topo IIα and Y656 in topo IIß inhibits these activities. Structural studies suggest that the differential impact of the CTD on topo IIα and topo IIß function may be due to differences in CTD charge distribution and differential alignment of the CTD with reference to transport DNA. Together these results suggest that topo IIα and topo IIß cooperate to maintain genome stability, which may be distinctly modulated by their CTDs.

Expression profile of COL2A1 and the pseudogene SLC6A10P predicts tumor recurrence in high-grade serous ovarian cancer.

Tumor recurrence, following initial response to adjuvant chemotherapy, is a major problem in women with high-grade serous ovarian cancer (HGSOC). Microarray analysis of primary tumors has identified genes that may be useful in risk stratification/overall survival, but are of limited value in predicting the >70% rate for tumor recurrence. In this study, we performed RNA-Seq analysis of primary and recurrent HGSOC to first identify unique differentially expressed genes. From this dataset, we selected 21 archetypical coding genes and one noncoding RNA, based on statistically significant differences in their expression profile between tumors, for validation by qPCR in a larger cohort of 110 ovarian tumors (71 primary and 39 recurrent) and for testing association of specific genes with time-to-recurrence (TTR). Kaplan-Meier tests revealed that high expression of collagen type II, alpha 1 (COL2A1) was associated with delayed TTR (HR = 0.47, 95% CI: 0.27-0.82, p = 0.008), whereas low expression of the pseudogene, solute carrier family 6 member 10 (SLC6A10P), was associated with longer TTR (HR = 0.53, 95% CI: 0.30-0.93, p = 0.027). Notably, TTR was significantly delayed for tumors that simultaneously highly expressed COL2A1 and lowly expressed SLC6A10P (HR = 0.21, 95% CI: 0.082-0.54, p = 0.0011), an estimated median of 95 months as compared to an estimated median of 16 months for subjects expressing other levels of COL2A1 and SLC6A10P. Thus, evaluating expression levels of COL2A1 and SLC6A10P at primary surgery could be beneficial for clinically managing recurrence of HGSOC.

Hydrogen sulfide modulates sinusoidal constriction and contributes to hepatic microcirculatory dysfunction during endotoxemia.

Hydrogen sulfide (H2S) affects vascular resistance; however, its effect on the hepatic microcirculation has not been investigated. Hepatic sinusoidal perfusion is dysregulated during sepsis, contributing to liver injury. Therefore, the present study determined the effect of H2S on the hepatic microcirculation and the contribution of endogenous H2S to hepatic microcirculatory dysfunction in an endotoxin model of sepsis. Portal infusion of H2S increased portal pressure in vivo (6.8 ± 0.2 mmHg before H2S vs. 8.6 ± 0.8 mmHg peak during H2S infusion, P < 0.05). Using intravital microscopy, we observed decreased sinusoidal diameter (6.2 ± 0.27 µm before H2S vs. 5.7 ± 0.3 µm after H2S, P < 0.05) and increased sinusoidal heterogeneity during H2S infusion (P < 0.05) and net constriction. Since hepatic H2S levels are elevated during sepsis, we used the cystathionine γ lyase inhibitor DL-propargylglycine (PAG) to determine the contribution of H2S to the hypersensitization of the sinusoid to the vasoconstrictor effect of endothelin-1 (ET-1). PAG treatment significantly attenuated the sinusoidal sensitization to ET-1 in endotoxin-treated animals. ET-1 infusion increased portal pressure to 175% of baseline in endotoxemic animals, which was reduced to 143% following PAG treatment (P < 0.05). PAG abrogated the increase in sinusoidal constriction after ET-1 infusion in LPS-treated rats (30.9% reduction in LPS rats vs. 11.6% in PAG/LPS rats, P < 0.05). Moreover, PAG treatment significantly attenuated the increase in NADH fluorescence following ET-1 exposure during endotoxemia (61 grayscale units LPS vs. 21 units in PAG/LPS, P < 0.05), suggesting an improvement in hepatic oxygen availability. This study is the first to demonstrate a vasoconstrictor action of H2S on the hepatic sinusoid and provides a possible mechanism for the protective effect of PAG treatment during sepsis.

Hydrogen sulfide differentially affects the hepatic vasculature in response to phenylephrine and endothelin 1 during endotoxemia.

Despite being protective in many disease states, hydrogen sulfide (H(2)S) contributes to organ injury in sepsis. Like the other gasotransmitters, nitric oxide and carbon monoxide, H(2)S is a modulator of the microcirculation. Because microcirculatory dysfunction is a main cause of organ injury during sepsis, the present study was designed to test the effect of H(2)S on microvascular dysfunction in isolated perfused livers. In most microcirculatory beds, endotoxin activates the endothelium, resulting in hyporesponsiveness to catecholamines and a derangement in blood flow distribution. We demonstrate that H(2)S treatment attenuates the increase in portal pressure during infusion of the α1 adrenergic agonist, phenylephrine (PE) (P < 0.01). Hydrogen sulfide almost completely negated the increase in portal pressure in livers isolated from endotoxemic rats. Treatment with an inhibitor of endogenous H(2)S, DL-propargylglycine (PAG), reversed lipopolysaccharide-induced hyporesponsiveness to PE. Because hepatic microcirculatory dysfunction is associated with excessive sinusoidal vasoconstriction and not dilation, we investigated whether H(2)S affects endothelin 1 (ET-1)-induced vasoconstriction in isolated livers. Contrary to PE treatment, H(2)S did not affect the increase in portal pressure during infusion of ET-1, nor did it attenuate the hypersensitization of the liver to ET-1 during endotoxemia. Hepatic resistance in control rats was increased by PAG treatment during ET-1 infusion, but this increase was not exacerbated during endotoxemia. We monitored hepatic O(2) consumption to assess the effect of vascular changes on oxygen consumption following ET-1 treatment. Low-dose ET-1 infusion caused an increase in hepatic O(2)consumption, whereas low-dose ET-1 infusion decreased O(2) consumption in endotoxemic livers. Interestingly, whereas we observed no effect of PAG on the vascular response to ET-1 infusion during endotoxemia, PAG treatment did maintain O(2), suggesting a more complex effect of H(2)S inhibition. In summary, the discrepancies between the hepatic response to PE and ET-1 suggest that H(2)S differentially contributes to microcirculatory dysfunction in the systemic and hepatic microcirculations. We propose that this is due to H(2)S exerting a differential vasoactive function on presinusoidal and sinusoidal sites within the liver. Moreover, our findings suggest that H(2)S may contribute to the progression of sepsis by contributing to microvascular failure.

The liver as a central regulator of hydrogen sulfide.

The liver is likely exposed to high levels of hydrogen sulfide (H2S) from endogenous hepatic synthesis and exogenous sources from the gastrointestinal tract. Little is known about the consequence of H2S exposure on the liver or hepatic regulation of H2S levels. We hypothesized that the liver has a high capacity to metabolize H2S and that H2S oxidation is decreased during sepsis, a condition in which hepatic O2 is limited and H2S synthesis is increased. Using a nonrecirculating isolated and perfused liver system, we demonstrated rapid hepatic H2S metabolism up to an infusion concentration of 200' µM H2S. Hydrogen sulfide metabolism was associated with an increase in O2 consumption from a baseline 96.7 ± 7.6 µmol O2/min/kg to 109 ± 7.4 µmol O2/min/kg at an infusion concentration of 150 µM H2S (P < 0.001). Removal of O2 from the perfusate decreased H2S clearance from a maximal 97% to only 23%. Livers isolated from rats subjected to cecal ligation and puncture (CLP) did not differ significantly from control livers in their capacity to metabolize H2S, suggesting that H2S oxidation remains a priority during sepsis. To test whether H2S induces O2 consumption in vivo, intravital microscopy was utilized to monitor the oxygen content in the hepatic microenvironment. Infusion of H2S increased the NADH/NAD+ ratio (645 gray-scale-unit increase, P = 0.035) and decreased hepatic O2 availability visualized with Ru(Phen)3(2+) (439 gray-scale-unit increase, P = 0.040). We conclude that the liver has a high hepatic capacity for H2S metabolism. Moreover, H2S oxidation consumes available oxygen and may exacerbate the tissue hypoxia associated with sepsis.