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Fold increase in myeloid cell subsets in the lungs.

CCR1 expression in tumor cells.

CCL23, the human CCL9 orthologous.

Myeloid cells, CCL9 and its effect on tumor phenotype.

Legend for Supplementary Figures S1-S6.

CCL9 promotes tumor cell survival and colonization.

CCL9 regulation by TGFβ-p38 pathway.

MYCN amplification (MNA) is a defining feature of high-risk neuroblastoma (NB) that predicts poor prognosis. However, whether genes within or in close proximity to the MYCN amplicon also contribute to aggressiveness in MNA+ NB remains poorly understood. Here we identify that GREB1, a transcription factor encoding gene neighboring the MYCN locus, is frequently co-expressed with MYCN, and promotes gene expression independently of MYCN in MNA+ NB. We found that GREB1, as a crucial estrogen receptor (ER) regulatory factor, is highly expressed in MNA+ NB comparable to ER+ breast cancer. Among GREB1-regulated genes altered in MNA+ NB, we uncover that Myosin 1B (MYO1B) promotes aggressive features in MNA+ NB, including invasive capacity in vitro, as well as extravasation and distant metastasis in vivo. Global secretome and proteome profiling uncover MYO1B as a major regulator of secretome reprogramming in MNA+ NB cells. Moreover, we identify the cytokine MIF as an important pro-invasive and pro-metastatic mediator of MYO1B activity. Together, we have identified a putative GREB1-MYO1B-MIF axis as an unconventional mechanism that promotes the aggressiveness of MNA+ NB, and independently of MYCN. Furthermore, we found that MYO1B is also upregulated in association with other oncoproteins during cellular transformation, and is dramatically increased in multiple human cancer types, suggesting a crucial role of MYO1B in cancers in addition to MNA+ NB. Citation Format: Hai-Feng Zhang, Alberto Delaidelli, Sumreen Javed, Busra Turgu, Christopher S. Hughes, Xiaqiu Yang, Michael Lizardo, Daniel Martinez, John M. Maris, Kristopher R. Bosse, Karla C. Williams, Poul H. Sorensen. A MYCN-independent mechanism mediating secretome reprogramming and metastasis in MYCN-amplified neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2593.

Ewing sarcoma (EwS) is a highly aggressive bone-associated tumor of children and young adults with strong propensity to progress and dismal outcome for patients with advanced stages of the disease. EwS is defined by a pathognomonic recurrent potent transcription factor, in 85-90% of cases EWSR1-FLI1. One of the previously described transcriptional targets of EWSR1-FLI1 is six-transmembrane epithelial antigen of the prostate 1 (STEAP1). STEAP1 is a member of the STEAP membrane-bound protein family involved in reduction of iron and copper which is a crucial step for metal transport and overexpressed in various malignancies. However, a precise functional role of STEAP1 in cancers including EwS remains elusive. EwS cell lines with knockout (KO) or knockdown (KD) of STEAP1 were created to perform functional studies for elucidating the functions of STEAP1 in EwS, including transient metal measurements. Additionally, the measurement of functional mitochondrial activity was performed by an extracellular flux analyzer. The activity of the mitochondrial electron transport chain complexes was measured by blue native PAGE. We showed that STEAP1 functions as a metalloreductase and STEAP1 KO cells have lower intracellular iron levels. Furthermore, we discovered that STEAP1 KO cells have significantly lower levels of the mitochondrial labile iron pool. Consequently, EwS cells with STEAP1 KO demonstrated impaired mitochondrial respiration and increased glycolysis rates. Additionally, the functional activity of iron-sulfur containing proteins and the the assembly of subunits of the mitochondrial electron transport chain complexes were decreased. This study is the first to demonstrate in vitro involvement of STEAP1 in transitional metal homeostasis and mitochondrial activity. These discoveries unveil alternative therapeutic strategies for EwS patients such as using compounds that promote an iron-dependent form of cell death, ferroptosis, or drugs that decrease metabolic adaptability. Citation Format: Taras Shyp, Rouhollah Mousavizadeh, Gian Luca Negri, Hai-Feng Zhang, Michael Lizardo, Poul Sorensen. STEAP1 facilitates iron transport in ewing sarcoma to support mitochondrial activity [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr B013.

Medulloblastoma (MB) is the most common pediatric brain malignancy, accounting for 20% of diagnosed brain tumors in children. The Group 3 (G3) MB subtype is found to be especially malignant and is commonly associated with MYC over-expression. Current G3 MB treatment regimens provide for 50% 5-year survival rates and are associated with severe side effects, including hearing loss and neurocognitive deficits. High expression of stress granule (SG) proteins such as G3BP1 and PAPBPC1 correlates with poor survival outcome in G3 MB patients. We propose that formation of SGs, mRNP granules assembled under cellular stress, is integral for MB tumor progression, and suggest that inhibition of SG formation is a novel therapeutic strategy. In order to identify key proteins for SG formation we set up a siRNA screen for 95 genes that have previously been linked to SG formation, and that are highly expressed in MB G3 tumors. The screen has been done in two G3 MB cell lines: MED8A and HDMB03. EIF2AK1, also known as HRI, was identified as one of the hits in the screen; it is an eIF2a kinase responsible for cellular response to oxidative stress. Phosphorylation of eIF2a is essential for canonical SG formation and global translation inhibition. We therefore used ISRIB, a known inhibitor of the integrated stress response, to block the effects of eIF2a phosphorylation, thus maintaining active global translation under stress conditions and preventing SG assembly. MB cells exposed to ISRIB combined with vincristine exhibited significantly decreased proliferation as well as increased apoptosis markers compared to cells exposed to vincristine alone. We show that combining SG inhibition with chemotherapy enhances the effects of the latter, which is increasingly relevant for patients suffering from therapy side effects and high chances of metastasis associated with G3 MB.

Metastases rarely occur in human livers with cirrhosis in clinical studies. We postulated that this phenomenon would also occur in experimental cirrhosis. Cirrhosis was established in C57BL/6 mice by carbon tetrachloride (CCl(4)) gastrogavage. B16F1 melanoma cells were injected into the mesenteric vein to induce hepatic metastases. Contrary to our postulate, there was greater than 4-fold increase in metastasis in animals with cirrhosis compared to controls. Intravital videomicroscopy showed that the hepatic sinusoids were narrower and more tumor cells were retained in the terminal portal vein (TPV) in cirrhotic livers. Immunohistochemistry demonstrated that the expression of vascular adhesion molecules was significantly increased in cirrhosis. Using confocal microscopy and the fluorescent nitric oxide (NO) probe 4,5-diaminofluorescein diacetate, a significantly lower level of NO release was detected in livers with cirrhosis both in basal conditions and after tumor cell arrest. Eight hours after mesenteric vein tumor cell injection, the percentage of apoptotic tumor cells in the sinusoids was 17% +/- 2% in livers with cirrhosis and 30% +/- 5% in normal livers. More mitotic and Ki-67 labeled tumor cells were seen in livers with cirrhosis. In conclusion, the changes in architecture and adhesion molecule expression in livers with cirrhosis may cause more tumor cells to arrest in the TPV. Lower levels of NO production may reduce apoptosis of B16F1 cells in livers with cirrhosis. As a result, these changes may promote the growth of metastasis in this cirrhotic model.

Background The human psoriasin (S100A7) gene has been implicated in inflammation and tumor progression. Implementation of a mouse model would facilitate further investigation of its function, however little is known of the murine psoriasin gene. In this study we have cloned the cDNA and characterized the expression of the potential murine ortholog of human S100A7/psoriasin in skin inflammation and mammary tumorigenesis. Methods On the basis of chromosomal location, phylogenetic analysis, amino acid sequence similarity, conservation of a putative Jab1-binding motif, and similarities of the patterns of mouse S100A7/psoriasin gene expression (measured by RT-PCR and in-situ hybridization) with those of human S100A7/psoriasin, we propose that mouse S100A7/psoriasin is the murine ortholog of human psoriasin/S100A7. Results Although mouse S100A7/psoriasin is poorly conserved relative to other S100 family members, its pattern of expression parallels that of the human psoriasin gene. In murine skin S100A7/psoriasin was significantly upregulated in relation to inflammation. In murine mammary gland expression is also upregulated in mammary tumors, where it is localized to areas of squamous differentiation. This mirrors the context of expression in human tumor types where both squamous and glandular differentiation occur, including cervical and lung carcinomas. Additionally, mouse S100A7/psoriasin possesses a putative Jab1 binding motif that mediates many downstream functions of the human S100A7 gene. Conclusion These observations and results support the hypothesis that the mouse S100A7 gene is structurally and functionally similar to human S100A7 and may offer a relevant model system for studying its normal biological function and putative role in tumor progression.