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Breast cancers demonstrate robust angiogenesis known as vascular niches despite an abundance of endogenous antiangiogenic factors in the tumor microenvironment. Aberrant angiogenesis may contribute to therapeutic resistance in antiangiogenic treatment via interactions between vascular endothelial cells (ECs) and cancer stem-like cells (CSCs, a subset of malignant cells critical for tumor initiation, metastasis and therapeutic resistance). Emerging evidence suggests that ECs in the tumor vasculature may confer CSC features to cancer cells by developing abnormal vascular niches. Protein kinase D1 (PKD1), a serine threonine kinase upstream of PI3Kinase/Akt and MAPK/Erk1/2, regulates angiogenesis and tumor progression. By analyzing RNA-sequencing data in the Cancer Genome Atlas (TCGA), we also found a poorer overall survival of the patients in the PKD1 high cohort compared with the patients with PKD1 low in BC tissues. Interestingly, our recent studies suggest that enhanced EC PKD1 signaling facilitates arteriolar niche formation for CSC self-renewal in breast cancers. However, the role and mechanisms of EC PKD-1 signaling in arteriolar niche establishment for CSC expansion in tumor progression is a critical yet underexplored and underappreciated field. We propose that EC PKD1 signaling in the vascular niches is essential for the expansion of CSCs to promote malignant progression of BCs. To test this hypothesis, we established a BC model in EC-specific PKD1 knockout mice and the control mice and developed a co-culture model of ECs and BC cells. By combining with molecular and cellular approaches and immunofluorescence microscopy, we observed that EC PKD1 signaling was critical for BC growth and survival of tumor-bearing mice, and particularly important in metastasis of BCs to the lungs. Analysis of the BC models indicated that there was an obvious enrichment of CSCs in the tumor vasculature of EC PKD1-positive mice. Moreover, there was a significant increase in expression of CD44 and ALDH1, the key CSC-related genes in BCs, in the primary tumors of mice with lung metastases. Co-culture of human microvascular ECs with human BC cells further confirmed that ECs promoted the expression of these two genes in tumor cells, along with the expression of other genes related to CSC self-renewal and epithelial mesenchymal transition. Co-culture with ECs also led to an increase in cell migration and colony formation in BCs. Importantly, the PKD1 signaling dictated the changes in CSC-related gene expression and aggressive behaviors in BC cells. In conclusion, EC PKD1 signaling in the vascular niches is critical for phenotype switching of BC cells to aggressive CSC features, thereby promoting BC progression and lung metastasis. Targeting PKD1-signaling-induced arteriolar niche establishment and this niche interaction with CSCs could provide potential novel and effective therapeutic strategies against tumor angiogenesis and CSC expansion. Citation Format: Abdellah Akil, Atul Kumar, Emily Spangler, Runhua Liu, Adam W. Beck, Lizhong Wang, Bassel El-Rayes, Bin Ren. Endothelial protein kinase D1 signaling induces development of aggressive cancer stem-like cells for breast cancer progression and lung metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB150.

Retrospective studies indicate that co-infection of hepatitis C virus (HCV) and human immunodeficiency virus (HIV) accelerates hepatic fibrosis progression. We have developed a co-culture system (MLH) comprising primary macrophages, hepatic stellate cells (HSC, LX-2), and hepatocytes (Huh-7), permissive for active replication of HCV and HIV, and assessed the effect of these viral infections on the phenotypic changes and fibrogenic gene expression in LX-2 cells. We detected distinct morphological changes in LX-2 cells within 24 hr post-infection with HCV, HIV or HCV/HIV in MLH co-cultures, with migration enhancement phenotypes. Human fibrosis microarrays conducted using LX-2 cell RNA derived from MLH co-culture conditions, with or without HCV and HIV infection, revealed novel insights regarding the roles of these viral infections on fibrogenic gene expression in LX-2 cells. We found that HIV mono-infection in MLH co-culture had no impact on fibrogenic gene expression in LX-2 cells. HCV infection of MLH co-culture resulted in upregulation (>1.9x) of five fibrogenic genes including CCL2, IL1A, IL1B, IL13RA2 and MMP1. These genes were upregulated by HCV/HIV co-infection but in a greater magnitude. Conclusion: Our results indicate that HIV-infected macrophages accelerate hepatic fibrosis during HCV/HIV co-infection by amplifying the expression of HCV-dependent fibrogenic genes in HSC.

Intrahepatic macrophages influence the composition of the microenvironment, host immune response to liver injury, and development of fibrosis. Compared with stellate cells, the role of macrophages in the development of fibrosis remains unclear. Multispectral imaging allows detection of multiple markers in situ in human formalin‐fixed, paraffin‐embedded tissue. This cutting‐edge technology is ideal for analyzing human liver tissues, as it allows spectral unmixing of fluorophore signals, subtraction of auto‐fluorescence, and preservation of hepatic architecture. We analyzed five different antibodies commonly observed on macrophage populations (CD68, MAC387, CD163, CD14, and CD16). After optimization of the monoplex stains and development of a Spectral Library, we combined all of the antibodies into a multiplex protocol and used them to stain biopsies collected from representative patients with chronic liver diseases, including chronic hepatitis C, nonalcoholic steatohepatitis, and autoimmune hepatitis. Various imaging modalities were tested, including cell phenotyping, tissue segmentation, t‐distributed stochastic neighbor embedding plots, and phenotype matrices that facilitated comparison and visualization of the identified macrophage and other cellular profiles. We then tested the feasibility of this platform to analyze numerous regions of interest from liver biopsies with multiple patients per group, using batch analysis algorithms. Five populations showed significant differences between patients positive for hepatitis C virus with advanced fibrosis when compared with controls. Three of these were significantly increased in patients with advanced fibrosis when compared to controls, and these included CD163+CD16+, CD68+, and CD68+MAC387+. Conclusion: Spectral imaging microscopy is a powerful tool that enables in situ analysis of macrophages and other cells in human liver biopsies and may lead to more personalized therapeutic approaches in the future., We optimized a spectral imaging microscopy platform to evaluate intrahepatic macrophages in liver biopsies from patients with chronic liver diseases (HCV, NASH, and AIH). We then compared differences in macrophage populations in patients with chronic viral hepatitis C and different stages of fibrosis (minimal and advanced), using batch analysis algorithms. Several macrophage phenotypes were significantly increased in patients with advanced fibrosis when compared to controls. Spectral imaging microscopy is a powerful tool that enables phenotypic characterization and quantification of intrahepatic macrophages, key players in hepatic fibrosis development that may be targets for future therapeutic intervention.

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Chromosomal instability (CIN) is frequently associated with a poor outcome in human carcinomas. The genomes of the main human malignancies are well defined as hundreds of tumors have been characterized by arrays. Targeting the appropriate chromosomes with set of markers appears as a realistic approach for CIN assessment. We decided to test the reliability of different insertion/deletion (InDel) polymorphisms to detect allelic loss in a subset of previously characterized hepatocellular carcinomas (HCC). To this aim 3 kinds of markers, L1 insertion (n=1), Alu insertions (n=4) and Marshfield InDel (MID, n=8) markers, were tested on a series of 68 paired HCC/non-tumor liver samples that were previously characterized for loss of heterozygosity (LOH). All markers were analyzed on agarose gels and some were tested with the high resolution melting (HRM) technique. Heterozygosity of the tested markers was high with a mean of 0.489 and a range of 0.265–0.525. Using 6 markers for chromosome 8p, the sensitivity of the method was high. LOH was detected in all samples known to be affected (n=34) whereas retention was found in 29/30 samples (specificity of 96.6%). Finally, the HRM analysis applied to 2 MID markers provided consistent profiles enabling closed-tube determination of chromosomes 17p and 18q status. Overall, our work suggests that different types of InDel markers are suitable for CIN detection in human tumors and may provide convenient and useful information for basic or translational research as well as for future applications in clinical practice.

Human immunodeficiency virus (HIV), hepatitis B virus (HBV) and hepatitis Cvirus (HCV) are major public health concerns. We aimed to determine the prevalence of HBV and HCV infections among HIV-infected patients, and to identify the main circulating hepatitis strains in Morocco. The study was carried out in 503 HIV-infected patients. Our survey indicated that the prevalence of HIV/hepatitis co-infection was 10.6%; 5.2% of patients were HBV surface antigen positive, and 5.4% of patients were anti-HCV positive. Among the HBV surface antigen-positive group, HBV DNA sequencing identified exclusively genotype D (D1: 26.7%; D7: 73.3%) in accordance with what is found in the general population. In contrast, sequencing of HCV isolates produced an unusual subtype distribution with a decreasing order of prevalence: 1a, 3a (both 23.5%), 1b, 4a (both 17.6%), 1c (11.8%) and 6h (6%).

International audience; Hepatocellular carcinoma is a major malignant tumor characterized in all areas by the disparity of risk between genders. The molecular bases of such disparity are still poorly understood. DNA-methyltransferase-3B (DNMT3B) may play an oncogenic role during tumorigenesis, and its genetic variants have been consistently associated with risk of several cancers, but a single study has investigated their roles in hepatocellular carcinoma (HCC). Polymorphisms of the DNMT3B gene may influence its activity on DNA methylation in several cancers, thereby modulating susceptibility to tumorigenesis. To test this hypothesis, we investigated the association between single nucleotide polymorphism -149C>T (rs2424913) in the promoter region DNMT3B and risk of HCC in a Moroccan population. In this case-control study, the DNMT3B SNP was genotyped by polymerase chain reaction-restriction fragment length polymorphism in 96 HCCs patients and 222 healthy controls that matched for age, sex and ethnicity. Overall, we found that, the DNMT3B 149 TT genotype was not significantly associated with increased risk of HCC (adjusted odds ratio (OR), 0.86, 95% CI, 0.41-1.80, P=0.697). Stratification analysis detected, however, a trend towards a profound risk in the female subset of patients (OR=2.04, 95% CI, 0.77-5.42) and a lesser risk for HCV-infected patients (OR=1.33, 95% CI, 0.43-4.17). Our findings contrast with those of previous studies performed in various cancers, which showed that individuals carrying at least one T allele have a significantly increased risk of developing cancer. In addition, we provide genetic evidence for the major difference of HCC risk between men and women. Further mechanistic studies are needed to unravel the underlying molecular mechanisms.

Infection by hepatitis C virus (HCV) is a major public-health problem. Chronic infection often leads to cirrhosis, steatosis, and hepatocellular carcinoma. The life cycle of HCV depends on the host cell machinery and involves intimate interaction between viral and host proteins. However, the role of host proteins in the life cycle of HCV remains poorly understood. Here, we identify the small ubiquitin-related modifier (SUMO1) as a key host factor required for HCV replication. We performed a series of cell biology and biochemistry experiments using the HCV JFH-1 (Japanese fulminate hepatitis 1) genotype 2a strain, which produces infectious particles and recapitulates all the steps of the HCV life cycle. We observed that SUMO1 is upregulated in Huh7.5 infected cells. Reciprocally, SUMO1 was found to regulate the expression of viral core protein. Moreover, knockdown of SUMO1 using specific siRNA influenced the accumulation of lipid droplets and reduced HCV replication as measured by qRT-PCR. Thus, we identify SUMO1 as a key host factor required for HCV replication. To our knowledge, this is the first report showing that SUMO1 regulates lipid droplets in the context of viral infection. Our report provides a meaningful insight into how HCV replicates and interacts with host proteins and is of significant importance for the field of HCV and RNA viruses.

International audience; Hepatitis C virus (HCV) isolates circulating in Morocco are poorly documented. To determine the subgenotype distribution of HCV in chronically infected patients, serum samples from 185 anti-HCV-positive patients were analyzed. Determination of the HCV genotype and subtype was performed by sequencing the 5'UTR, NS5B and core regions. According to the NS5B phylogeny, the HCV strains primarily belonged to subtypes 1b (75.2%), 2i (19.1%) and 2k (2.8%). Using a Bayesian approach, the mean date of appearance of the most recent common ancestor was estimated to be 1910 for HCV-1b and 1854 for HCV-2i. Although it is currently the most frequent genotype in Morocco and the dominant form in hepatocellular carcinoma, it thus appears that HCV-1b was introduced into the population subsequently to HCV-2i.

Background and aims : Hepatocellular carcinoma (HCC) is a common cause of death by cancer worldwide. In Morocco, HCC is characterized by few mutations and a mild chromosome instability suggesting that epigenetic changes may represent the driving force of tumorigenesis in the region. Recently, three studies looked for an association between EP300 or PCAF polymorphisms and cancer but there is a conspicuous lack of data regarding these histone acetyltransferase (HAT) variants and HCC development. The aim of the current study was to assess the impact of the Ile997Val in EP300 and Asn386Ser in PCAF polymorphisms on the risk of HCC. Materials and methods : We performed a case–control study comparing 94 cases with HCC and 220 matching controls. Sequencing methods were used to determine the genotype at the Ile997Val and Asn386Ser on EP300 and PCAF . Results : We found an overall association between genotypes Val/Val in EP300 and HCC risk (OR, 3.03; 95% CI, 1.08–8.47; P = 0.028). Population stratifications revealed a trend or significantly higher risks of HCC development for women and HCV-negative patients carrying the EP300 Val/Val genotype (OR, 4.06; 95% CI, 0.71–23.36; P = 0.09 and OR, 4.48; 95% CI, 1.04–19.14; P = 0.02, respectively). The PCAF Ser/Ser genotype at codon 386 was more frequent in HCC cases than in control group ( P = 0.03). We observed trends for higher risk of HCC among men and/or HCV-negative patients carrying Ser/Ser genotype when compared with controls (OR, 10.62; 95% CI, 0.50–225.13 and OR, 11.78; 95% CI, 0.47–295.56, respectively). Conclusion : It appears that variants of the transcriptional coactivator genes ( EP300 and PCAF ) may influence HCC risk in populations with low mutations or chromosomal instability rates. Additional surveys are warranted to confirm this first report.