Post-translational prenylation mechanisms, including farnesylation and geranylgeranylation, mediate both subcellular localization and protein-protein interaction in eukaryotes. The prenyltransferase complex is an αβ heterodimer in which the essential α-subunit is common to both the farnesyltransferase and the geranylgeranyltransferase type-I enzymes. The β-subunit is unique to each enzyme. Farnesyltransferase activity is an important mediator of protein localization and subsequent signaling for multiple proteins, including Ras GTPases. Here, we examined the importance of protein farnesylation in the opportunistic fungal pathogen Aspergillus fumigatus through generation of a mutant lacking the farnesyltransferase β-subunit, ramA. Although farnesyltransferase activity was found to be non-essential in A. fumigatus, diminished hyphal outgrowth, delayed polarization kinetics, decreased conidial viability, and irregular distribution of nuclei during polarized growth were noted upon ramA deletion (ΔramA). Although predicted to be a target of the farnesyltransferase enzyme complex, we found that localization of the major A. fumigatus Ras GTPase protein, RasA, was only partially regulated by farnesyltransferase activity. Furthermore, the farnesyltransferase-deficient mutant exhibited attenuated virulence in a murine model of invasive aspergillosis, characterized by decreased tissue invasion and development of large, swollen hyphae in vivo. However, loss of ramA also led to a Cyp51A/B-independent increase in resistance to triazole antifungal drugs. Our findings indicate that protein farnesylation underpins multiple cellular processes in A. fumigatus, likely due to the large body of proteins affected by ramA deletion.

Pathogenic fungi employ numerous mechanisms to flourish in the stressful environment encountered within their mammalian hosts. Central to this arsenal for filamentous fungi is invasive growth within the host microenvironment, mediated by establishment and maintenance of polarized hyphal morphogenesis. In Aspergillus fumigatus, the RasA signal transduction pathway has emerged as a significant regulator of hyphal morphogenesis and virulence, among other processes. The factors contributing to the regulation of RasA itself are not as thoroughly understood, although proper temporal activation of RasA and spatial localization of RasA to the plasma membrane are known to play major roles. Interference with RasA palmitoylation or prenylation results in mislocalization of RasA and is associated with severe growth deficits. In addition, dysregulation of RasA activation results in severe morphologic aberrancies and growth deficits. This review highlights the relationship between RasA signaling, hyphal morphogenesis, and virulence in Aspergillus fumigatus, and focuses on potential determinants of spatial and temporal RasA regulation.

Aspergillus fumigatus is an important fungal pathogen against which limited treatments exist. During invasive disease, A. fumigatus hyphae grow in a highly polarized fashion, forming filaments that invade blood vessels and disseminate to distant sites. Once invasion and dissemination occur, mortality rates are high. We have previously shown that the Ras signaling pathway is an important regulator of the hyphal growth machinery supporting virulence in A. fumigatus. Here, we show that functional Ras signaling in A. fumigatus requires a novel, fungus-specific domain within the Ras protein. This domain is highly conserved among fungi, yet absent in higher eukaryotes, suggesting a potentially crucial difference in the regulation of Ras pathway activity between the human host and the fungal pathogen. Exploration of the mechanisms through which this domain regulates signaling could lead to novel antifungal therapies specifically targeting fungal Ras pathways.

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B7-H3 (CD276) is a cell surface glycoprotein of B7 family of checkpoint blockade proteins that has been considered a valuable novel biomarker and a promising target for cancer treatment. B7-H3 is overexpressed in a wide variety of human cancers and is associated with tumor progression, metastasis, and poor patient outcome. In addition to its function in cancer immunity, B7-H3 also has tumor-intrinsic functions related to drug resistance, metabolic reprogramming, migration, and invasion of cancer cells. B7-H3 overexpression in the ovarian tumors and tumor vasculature has been reported and B7-H3 monoclonal antibodies have shown inhibitory effects in ovarian cancer xenograft studies. Nonetheless, the molecular pathways regulated by B7-H3 in ovarian cancer remain largely unknown. Here we explored the role of tumor intrinsic B7-H3 in ovarian cancer cells by genetic modulation of B7-H3 expression levels in ovarian cancer cells, using CRISPR/Cas9-mediated B7-H3 knockout and B7-H3 overexpression using retrovirus system for cell transduction. Knockout of B7-H3 in OV-90 cells, which showed high endogenous expression of B7-H3, resulted in decreased glycolysis and oxidative phosphorylation through downregulation of HIF1α, and its downstream target glycolytic proteins LDHA and PDK1. Furthermore, OV-90 B7-H3 KO cells showed decreased phosphorylation of the prosurvival protein STAT3, reduced clonogenic and migratory capability, and increased sensitivity to chemotherapeutic agent carboplatin. Overexpression of B7-H3 in TOV112D cells, which has low endogenous expression of B7-H3, resulted in the opposite effects, leading to increased glycolysis and mitochondrial respiration, increased STAT3 phosphorylation, increased migration, and increased resistance to carboplatin. We also performed RNA-seq analysis of OV-90 B7-H3 WT and KO clones and found several signaling pathways involved in cell migration, adhesion, and inflammatory response. Finally, we found that the growth of OV-90 B7-H3 KO cells was dramatically defective in xenograft models using both athymic nude and SCID immunocompromised mice. These results suggest that tumor-intrinsic B7-H3 plays a critical role in the growth of ovarian cancer and warrants further investigation of therapeutic approaches blocking B7-H3 functions for the treatment of this deadly malignancy in women. Citation Format: Holly Taylor, Jaroslav Slamecka, Alla Musiyenko, Elaine Gavin, Tiffany S. Norton, Ileana Aragon, Taylor Young, Jennifer Scalici, Rodney P. Rocconi, Ming Tan, Luciana Madeira da Silva. Tumor-intrinsic B7-H3 regulates drug resistance, metabolism, and pathogenesis in ovarian cancer. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B31.