Triple-negative breast cancer (TNBC) is cancer that tested as negative for estrogen receptors (ER), progesterone receptors (PR), and excess human epidermal growth factor receptor 2 (HER2) protein which accounts for 15%–20% of all breast cancer cases. TNBC is considered to be a poorer prognosis than other types of breast cancer, mainly because it involves more aggressive phenotypes that are similar to stem cell–like cancer cells (cancer stem cell, CSC). Thus, targeted treatment of TNBC remains a major challenge in clinical practice. This review article surveys the latest evidence concerning the role of genomic alteration in current TNBC treatment responses, current clinical trials and potential targeting sites, CSC and drug resistance, and potential strategies targeting CSCs in TNBC. Furthermore, the role of insulin-like growth factor 1 receptor (IGF-1R) and nicotinic acetylcholine receptors (nAChR) in stemness expression, chemoresistance, and metastasis in TNBC and their relevance to potential treatments are also discussed and highlighted.

Septins are members of the GTPase superfamily, which has been implicated in diverse cellular functions including cytokinesis and morphogenesis. Septin 12 (SEPT12) is a testis-specific gene critical for the terminal differentiation of male germ cells. We report the identification of two missense SEPT12 mutations, c.266C>T/p.Thr89Met and c.589G>A/p.Asp197Asn, in infertile men. Both mutations are located inside the GTPase domain and may alter the protein structure as suggested by in silico modeling. The p.Thr89Met mutation significantly reduced guanosine-5′-triphosphate (GTP) hydrolytic activity, and the p.Asp197Asn mutation (SEPT12^D197N) interfered with GTP binding. Both mutant SEPT12 proteins restricted the filament formation of the wild-type SEPT12 in a dose-dependent manner. The patient carrying SEPT12^D197N presented with oligoasthenozoospermia, whereas the SEPT12^T89M patient had asthenoteratozoospermia. The characteristic sperm pathology of the SEPT12^D197N patient included defective annulus with bent tail and loss of SEPT12 from the annulus of abnormal sperm. Our finding suggests loss-of-function mutations in SEPT12 disrupted sperm structural integrity by perturbing septin filament formation. Hum Mutat 33:710–719, 2012.

Background: The inflammatory cytokine interleukin-6 (IL-6) is critical for the expression of octamer-binding transcription factor 4 (OCT4), which is highly associated with early tumor recurrence and poor prognosis of hepatocellular carcinomas (HCC). DNA methyltransferase (DNMT) family is closely linked with OCT4 expression and drug resistance. However, the underlying mechanism regarding the interplay between DNMTs and IL-6-induced OCT4 expression and the sorafenib resistance of HCC remains largely unclear.Methods: HCC tissue samples were used to examine the association between DNMTs/OCT4 expression levels and clinical prognosis. Serum levels of IL-6 were detected using ELISA assays (n = 144). Gain- and loss-of-function experiments were performed in cell lines and mouse xenograft models to determine the underlying mechanism in vitro and in vivo.Results: We demonstrate that levels of DNA methyltransferase 3 beta (DNMT3b) are significantly correlated with the OCT4 levels in HCC tissues (n = 144), and the OCT4 expression levels are positively associated with the serum IL-6 levels. Higher levels of IL-6, DNMT3b, or OCT4 predicted early HCC recurrence and poor prognosis. We show that IL-6/STAT3 activation increases DNMT3b/1 and OCT4 in HCC. Activated phospho-STAT3 (STAT-Y640F) significantly increased DNMT3b/OCT4, while dominant negative phospho-STAT3 (STAT-Y705F) was suppressive. Inhibiting DNMT3b with RNA interference or nanaomycin A (a selective DNMT3b inhibitor) effectively suppressed the IL-6 or STAT-Y640F-induced increase of DNMT3b-OCT4 and ALDH activity in vitro and in vivo. The fact that OCT4 regulates the DNMT1 expressions were further demonstrated either by OCT4 forced expression or DNMT1 silence. Additionally, the DNMT3b silencing reduced the OCT4 expression in sorafenib-resistant Hep3B cells with or without IL-6 treatment. Notably, targeting DNMT3b with nanaomycin A significantly increased the cell sensitivity to sorafenib, with a synergistic combination index (CI) in sorafenib-resistant Hep3B cells.Conclusions: The DNMT3b plays a critical role in the IL-6-mediated OCT4 expression and the drug sensitivity of sorafenib-resistant HCC. The p-STAT3 activation increases the DNMT3b/OCT4 which confers the tumor early recurrence and poor prognosis of HCC patients. Findings from this study highlight the significance of IL-6-DNMT3b–mediated OCT4 expressions in future therapeutic target for patients expressing cancer stemness-related properties or sorafenib resistance in HCC.

Male infertility has become a worldwide health problem, but the etiologies of most cases are still unknown. SEPT12, a GTP-binding protein, is involved in male fertility. Two SEPT12 mutations (SEPT12^T89M and SEPT12^D197N) have been identified in infertile men who have a defective sperm annulus with a bent tail. The function of SEPT12 in the sperm annulus is still unclear. Here, we found that SEPT12 formed a filamentous structure with SEPT7, SEPT 6, SEPT2 and SEPT4 at the sperm annulus. The SEPT12-based septin core complex was assembled as octameric filaments comprising the SEPT proteins 12-7-6-2-2-6-7-12 or 12-7-6-4-4-6-7-12. In addition, the GTP-binding domain of SEPT12 was crucial for its interaction with SEPT7, and the N- and C-termini of SEPT12 were required for the interaction of SEPT12 with itself to polymerize octamers into filaments. Mutant mice carrying the SEPT12^D197N mutation, which disrupts SEPT12 filament formation, showed a disorganized sperm annulus, bent tail, reduced motility and loss of the SEPT ring structure at the sperm annulus. These phenotypes were also observed in an infertile man carrying SEPT12^D197N. Taken together, our results demonstrate the molecular architecture of SEPT12 filaments at the sperm annulus, their mechanical support of sperm motility, and their correlation with male infertility.

Septins (full name: Septin; symbol name: SEPT) belong to a family of polymerizing GTP binding proteins that are required for many cellular functions, including membrane compartmentalization, vesicl...

Septins are critical for numerous cellular processes through the formation of heteromeric filaments and rings indicating the importance of structural regulators in septin assembly. Several posttranslational modifications (PTMs) mediate the dynamics of septin filaments in yeast. However, little is known about the role of PTMs in regulating mammalian septin assembly, and the in vivo significance of PTMs on mammalian septin assembly and function remains unknown. Here, we showed that SEPT12 was phosphorylated on Ser198 using mass spectrometry, and we generated SEPT12 phosphomimetic knock-in (KI) mice to study its biological significance. The homozygous KI mice displayed poor male fertility due to deformed sperm with defective motility and loss of annulus, a septin-based ring structure. Immunohistochemistry of KI testicular sections suggested that SEPT12 phosphorylation inhibits septin ring assembly during annulus biogenesis. We also observed that SEPT12 was phosphorylated via PKA, and its phosphorylation interfered with SEPT12 polymerization into complexes and filaments. Collectively, our data indicate that SEPT12 phosphorylation inhibits SEPT12 filament formation, leading to loss of the sperm annulus/septin ring and poor male fertility. Thus, we provide the first in vivo genetic evidence characterizing importance of septin phosphorylation in the assembly, cellular function and physiological significance of septins. Septin polymerization into filaments and rings are built for numerous cellular processes, and misregulation of septin filaments is implicated in several human diseases. Therefore, it is important to identify structural regulator of septin assembly. We showed that SEPT12 was phosphorylated on the Ser198 residue, and phosphomimetic SEPT12 KI mice display defective male fertility, impaired sperm motility and loss of septin-organized sperm annulus. Homozygous KI spermatids exhibited complete absence of septin ring assembly during annulus biogenesis. Additionally, SEPT12 phosphorylation through PKA interfered with SEPT12 polymerization into complexes and filaments. Thus, SEPT12 phosphorylation regulates SEPT12 polymerization that is involved in the septin ring assembly/sperm annulus establishment, the structural and mechanical support of sperm and robust male fertility. The present study provides novel insight into spermatogenetic regulation, highlights the importance of phosphorylation as structural regulator of septin assembly in vivo, and identifies the mechanism by which phosphorylation contributes to septin assembly.