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The cytochrome b5 domain containing 2 (CYB5D2; Neuferricin) protein has been reported to bind heme, however, the critical residues responsible for heme-binding are undefined. Furthermore, the relationship between heme-binding and CYB5D2-mediated intracellular functions remains unknown. Previous studies examining heme-binding in two cytochrome b5 heme-binding domain-containing proteins, damage-associated protein 1 (Dap1; Saccharomyces cerevisiae) and human progesterone receptor membrane component 1 (PGRMC1), have revealed that conserved tyrosine (Y) 73, Y79, aspartic acid (D) 86, and Y127 residues present in human CYB5D2 may be involved in heme-binding. CYB5D2 binds to type b heme, however, only the substitution of glycine (G) at D86 (D86G) within its cytochrome b5 heme-binding (cyt-b5) domain abolished its heme-binding ability. Both CYB5D2 and CYB5D2(D86G) localize to the endoplasmic reticulum. Ectopic CYB5D2 expression inhibited cell proliferation and anchorage-independent colony growth of HeLa cells. Conversely, CYB5D2 knockdown and ectopic CYB5D2(D86G) expression increased cell proliferation and colony growth. As PGRMC1 has been reported to regulate the expression and activities of cytochrome P450 proteins (CYPs), we examined the role of CYB5D2 in regulating the activities of CYPs involved in sterol synthesis (CYP51A1) and drug metabolism (CYP3A4). CYB5D2 co-localizes with cytochrome P450 reductase (CYPOR), while CYB5D2 knockdown reduced lanosterol demethylase (CYP51A1) levels and rendered HeLa cells sensitive to mevalonate. Additionally, knockdown of CYB5D2 reduced CYP3A4 activity. Lastly, CYB5D2 expression conferred HeLa cell survival from chemotherapeutic agents (paclitaxel, cisplatin and doxorubicin), with its ability to promote survival being dependent on its heme-binding ability. Taken together, this study provides evidence that heme-binding is critical for CYB5D2 in regulating HeLa cell growth and survival, with endogenous CYB5D2 being required to modulate CYP activities. [ABSTRACT FROM AUTHOR]

Prostate cancer stem-like cells (PCSCs) are being intensely investigated largely owing to their contributions towards prostate tumorigenesis, however, our understanding of PCSC biology, including their critical pathways, remains incompletely understood. While epidermal growth factor (EGF) is widely used in maintaining PCSC cells in vitro, the importance of EGF-dependent signaling and its downstream pathways in PCSC self-renewal are not well characterized. By investigating DU145 sphere cells, a population of prostate cancer cells with stem-like properties, we report here that epidermal growth factor receptor (EGFR) signaling plays a critical role in the propagation of DU145 PCSCs. Activation of EGFR signaling via addition of EGF and ectopic expression of a constitutively-active EGFR mutant (EGFRvIII) increased sphere formation. Conversely, inhibition of EGFR signaling by using EGFR inhibitors (AG1478 and PD168393) and knockdown of EGFR significantly inhibited PCSC self-renewal. Consistent with the MEK-ERK pathway being a major target of EGFR signaling, activation of the MEK-ERK pathway contributed to EGFR-facilitated PCSC propagation. Modulation of EGFR signaling affected extracellular signal-related kinase (ERK) activation. Inhibition of ERK activation through multiple approaches, including treatment with the MEK inhibitor U0126, ectopic expression of dominant-negative MEK1(K97M), and knockdown of either ERK1 or ERK2 resulted in a robust reduction in PCSC propagation. Collectively, the present study provides evidence that EGFR signaling promotes PCSC self-renewal, in part, by activating the MEK-ERK pathway. [ABSTRACT FROM AUTHOR]