Emerging Role of SOX Proteins in Breast Cancer Development and Maintenance.

The SOX genes encode a family of more than 20 transcription factors that are critical regulators of embryogenesis and developmental processes and, when aberrantly expressed, have been shown to contribute to tumor development and progression in both an oncogenic and tumor suppressive role. Increasing evidence demonstrates that the SOX proteins play essential roles in multiple cellular processes that mediate or contribute to oncogenic transformation and tumor progression. In the context of breast cancer, SOX proteins function both as oncogenes and tumor suppressors and have been shown to be associated with tumor stage and grade and poor prognosis. Experimental evidence demonstrates that a subset of SOX proteins regulate critical aspects of breast cancer biology including cancer stemness and multiple signaling pathways leading to altered cell proliferation, survival, and tumor development; EMT, cell migration and metastasis; as well as other tumor associated characteristics. This review will summarize the role of SOX family members as important mediators of tumorigenesis in breast cancer, with an emphasis on the triple negative or basal-like subtype of breast cancer, as well as examine the therapeutic potential of these genes and their downstream targets.

Determinants of pore folding in potassium channel biogenesis.

Many ion channels, both selective and nonselective, have reentrant pore loops that contribute to the architecture of the permeation pathway. It is a fundamental feature of these diverse channels, regardless of whether they are gated by changes of membrane potential or by neurotransmitters, and is critical to function of the channel. Misfolding of the pore loop leads to loss of trafficking and expression of these channels on the cell surface. Mature tetrameric potassium channels contain an α-helix within the pore loop. We systematically mutated the "pore helix" residues of the channel Kv1.3 and assessed the ability of the monomer to fold into a tertiary reentrant loop. Our results show that pore loop residues form a canonical α-helix in the monomer early in biogenesis and that disruption of tertiary folding is caused by hydrophilic substitutions only along one face of this α-helix. These results provide insight into the determinants of the reentrant pore conformation, which is essential for ion channel function.

IL-10-producing regulatory B cells in the pathogenesis of chronic hepatitis B virus infection.

A regulatory subset of B cells has been found to modulate immune responses in autoimmunity, infection, and cancer, but it has not been investigated in the setting of human persistent viral infection. IL-10 is elevated in patients with chronic hepatitis B virus infection (CHB), but its cellular sources and impact on antiviral T cells have not been addressed. We investigated the role of IL-10 and regulatory B cells in the pathogenesis of CHB. Serum IL-10 levels were studied longitudinally in patients with CHB undergoing spontaneous disease flares. There was a close temporal correlation between IL-10 levels and fluctuations in viral load or liver inflammation. Blockade of IL-10 in vitro rescued polyfunctional virus-specific CD8 T cell responses. To investigate the potential contribution of regulatory B cells, their frequency was measured directly ex vivo and after exposure to stimuli relevant to hepatitis B virus (HBV) (CpG or HBV Ags). IL-10-producing B cells were enriched in patients, and their frequency correlated temporally with hepatic flares, both after stimulation and directly ex vivo. Phenotypically, these cells were predominantly immature (CD19(+)CD24(hi)CD38(hi)) ex vivo; sorted CD19(+)CD24(hi)CD38(hi) cells suppressed HBV-specific CD8 T cell responses in an IL-10-dependent manner. In summary, these data reveal a novel IL-10-producing subset of B cells able to regulate T cell immunity in CHB.

Cidofovir in Severe Hypoxemic Adenoviral Pneumonia.

The authors present a 16-mo-old boy with flu like symptoms, not responding to supportive management and progressed to severe hypoxemic pneumonia. Adenovirus was detected in the nasopharyngeal aspirate. He showed rapid improvement after intravenous cidofovir administration.

Development and Validation of a Cell-Based Binding Neutralizing Antibody Assay for an Antibody-Drug Conjugate.

The utilization of antibody-drug conjugates (ADCs) has gained considerable attention in the field of targeted cancer therapy due to their ability to synergistically combine the specificity of monoclonal antibodies (mAbs) and the potency of small molecular drugs. However, the immunogenic nature of the antibody component within ADCs warrants the need for robust immunogenicity testing, including a neutralizing antibody (NAb) assay. Since the mechanism of action (MOA) of the ADC is to first bind to the target cells and then release the payload intracellularly to kill the cells, the most relevant NAb assay format would be a cell-based killing assay. However, in this paper, we present a case where a cell-based killing assay could not be developed after multiple cell lines and NAb-positive controls (PC) had been tested. Surprisingly, contrary to our expectations, all NAb PCs tested exhibited an increased killing effect on the target cells, instead of the expected protective response. This unexpected phenomenon most likely is due to the non-specific internalization of drug/NAb complexes via FcγRs, as an excessive amount of human IgG1 and mouse IgG2a, but not mouse IgG1, greatly inhibited drug or drug/NAb complexes induced cell death. To overcome this obstacle, we implemented a novel cell-based binding assay utilizing the Meso Scale Discovery (MSD) platform. We also propose that an in vitro cell killing NAb assay is limited to at best monitoring the target binding and internalization induced cell death, but not by-stander killing induced by prematurely released or dead-cell released payload, hence cannot really mimic the in vivo MOA of ADC.

SOX4-SMARCA4 complex promotes glycolysis-dependent TNBC cell growth through transcriptional regulation of Hexokinase 2.

Tumor cells rely on increased glycolytic capacity to promote cell growth and progression. While glycolysis is known to be upregulated in the majority of triple negative (TNBC) or basal-like subtype breast cancers, the mechanism remains unclear. Here, we used integrative genomic analyses to identify a subset of basal-like tumors characterized by increased expression of the oncogenic transcription factor SOX4 and its co-factor the SWI/SNF ATPase SMARCA4. These tumors are defined by unique gene expression programs that correspond with increased tumor proliferation and activation of key metabolic pathways, including glycolysis. Mechanistically, we demonstrate that the SOX4-SMARCA4 complex mediates glycolysis through direct transcriptional regulation of Hexokinase 2 (HK2) and that aberrant HK2 expression and altered glycolytic capacity are required to mediate SOX4-SMARCA4-dependent cell growth. Collectively, we have defined the SOX4-SMARCA4-HK2 signaling axis in basal-like breast tumors and established that this axis promotes metabolic reprogramming which is required to maintain tumor cell growth.

Blockade of immunosuppressive cytokines restores NK cell antiviral function in chronic hepatitis B virus infection.

NK cells are enriched in the liver, constituting around a third of intrahepatic lymphocytes. We have previously demonstrated that they upregulate the death ligand TRAIL in patients with chronic hepatitis B virus infection (CHB), allowing them to kill hepatocytes bearing TRAIL receptors. In this study we investigated whether, in addition to their pathogenic role, NK cells have antiviral potential in CHB. We characterised NK cell subsets and effector function in 64 patients with CHB compared to 31 healthy controls. We found that, in contrast to their upregulated TRAIL expression and maintenance of cytolytic function, NK cells had a markedly impaired capacity to produce IFN-γ in CHB. This functional dichotomy of NK cells could be recapitulated in vitro by exposure to the immunosuppressive cytokine IL-10, which was induced in patients with active CHB. IL-10 selectively suppressed NK cell IFN-γ production without altering cytotoxicity or death ligand expression. Potent antiviral therapy reduced TRAIL-expressing CD56(bright) NK cells, consistent with the reduction in liver inflammation it induced; however, it was not able to normalise IL-10 levels or the capacity of NK cells to produce the antiviral cytokine IFN-γ. Blockade of IL-10 +/- TGF-ß restored the capacity of NK cells from both the periphery and liver of patients with CHB to produce IFN-γ, thereby enhancing their non-cytolytic antiviral capacity. In conclusion, NK cells may be driven to a state of partial functional tolerance by the immunosuppressive cytokine environment in CHB. Their defective capacity to produce the antiviral cytokine IFN-γ persists in patients on antiviral therapy but can be corrected in vitro by IL-10+/- TGF-ß blockade.

Role of the coinhibitory receptor cytotoxic T lymphocyte antigen-4 on apoptosis-Prone CD8 T cells in persistent hepatitis B virus infection.

UNLABELLED: An excess of coinhibitory signals has been proposed to drive the T-cell exhaustion characteristic of persistent viral infections. In this study we examined the contribution of the coinhibitory receptor cytotoxic T lymphocyte antigen-4 (CTLA-4) to CD8 T cell tolerance in chronic hepatitis B virus (HBV) infection (CHB). CD8 T cells in patients with CHB have an increased propensity to express the coinhibitory receptor CTLA-4 and this correlates with viral load. CTLA-4 is up-regulated on those HBV-specific CD8 T cells with the highest levels of the proapoptotic protein Bim, which we have previously shown mediates their premature attrition; abrogation of CTLA-4-mediated coinhibition can reduce Bim expression. Longitudinal study of CHB patients beginning antiviral therapy reveals that HBV DNA suppression induces transient reconstitution of HBV-specific CD8 T cells but does not reprogram their CTLA-4(hi) Bim(hi) tolerogenic phenotype. Blocking CTLA-4 is able to increase the expansion of interferon gamma (IFN-γ)-producing HBV-specific CD8 T cells in both the peripheral and intrahepatic compartments. The rescue of anti-HBV responses by either CTLA-4 or PD-L1 blockade is nonredundant. CONCLUSION: CTLA-4 is expressed by HBV-specific CD8 T cells with high levels of Bim and helps to drive this proapoptotic phenotype. CTLA-4 blockade could form one arm of a therapeutic approach to modulate the diverse patterns of coregulation of T-cell exhaustion in this heterogeneous disease.

Nuclear Receptor-Mediated Metabolic Reprogramming and the Impact on HR+ Breast Cancer.

Metabolic reprogramming enables cancer cells to adapt to the changing microenvironment in order to maintain metabolic energy and to provide the necessary biological macromolecules required for cell growth and tumor progression. While changes in tumor metabolism have been long recognized as a hallmark of cancer, recent advances have begun to delineate the mechanisms that modulate metabolic pathways and the consequence of altered signaling on tumorigenesis. This is particularly evident in hormone receptor positive (HR+) breast cancers which account for approximately 70% of breast cancer cases. Emerging evidence indicates that HR+ breast tumors are dependent on multiple metabolic processes for tumor progression, metastasis, and therapeutic resistance and that changes in metabolic programs are driven, in part, by a number of key nuclear receptors including hormone-dependent signaling. In this review, we discuss the mechanisms and impact of hormone receptor mediated metabolic reprogramming on HR+ breast cancer genesis and progression as well as the therapeutic implications of these metabolic processes in this disease.

SOX4 and SMARCA4 cooperatively regulate PI3k signaling through transcriptional activation of TGFBR2.

Dysregulation of PI3K/Akt signaling is a dominant feature in basal-like or triple-negative breast cancers (TNBC). However, the mechanisms regulating this pathway are largely unknown in this subset of aggressive tumors. Here we demonstrate that the transcription factor SOX4 is a key regulator of PI3K signaling in TNBC. Genomic and proteomic analyses coupled with mechanistic studies identified TGFBR2 as a direct transcriptional target of SOX4 and demonstrated that TGFBR2 is required to mediate SOX4-dependent PI3K signaling. We further report that SOX4 and the SWI/SNF ATPase SMARCA4, which are uniformly overexpressed in basal-like tumors, form a previously unreported complex that is required to maintain an open chromatin conformation at the TGFBR2 regulatory regions in order to mediate TGFBR2 expression and PI3K signaling. Collectively, our findings delineate the mechanism by which SOX4 and SMARCA4 cooperatively regulate PI3K/Akt signaling and suggest that this complex may play an essential role in TNBC genesis and/or progression.

Temporal analysis of early immune responses in patients with acute hepatitis B virus infection.

BACKGROUND & AIMS: Hepatitis B virus (HBV) causes more than 1 million deaths annually from immune-mediated liver damage. The long incubation period has been difficult to study; by the time most patients present, massive viremia and the majority of viral clearance have already occurred. The aim of this study was to investigate the contribution of innate and adaptive immune mechanisms in early acute HBV through access to an unusual cohort of patients sampled in the preclinical phase and followed up to resolution of their infection. METHODS: Twenty-one patients with acute HBV were studied, 8 of them from before the peak of viremia. Circulating innate cytokines were quantitated by enzyme-linked immunosorbent assay and natural killer (NK) and T-cell effector function by flow cytometry. Results were correlated with temporal changes in viral load, serology, and liver inflammation and compared with healthy controls. RESULTS: Type I interferon (IFN) remained barely detectable throughout, with concentrations no higher than those found in healthy controls. Similarly, interleukin-15 and IFN-lambda1 were not induced during peak viremia. NK cell activation and capacity for IFN-gamma production were reduced at peak viremia. Early functional HBV-specific CD4 and CD8 T-cell responses were attenuated as viral load increased and recovered again as infection resolved. The transient inhibition of NK and T-cell responses coincided with a surge in the immunosuppressive cytokine interleukin-10 accompanying HBV viremia. CONCLUSIONS: The early stages of acute HBV are characterized by induction of interleukin-10 rather than type I IFN, accompanied by a temporary attenuation of NK and T-cell responses.

Structural and immunogenic effects of multiple hapten loading on carrier protein.

Haptens are low-molecular-weight compounds that are usually nonimmunogenic in nature. These compounds are, in general, conjugated with carrier proteins to elicit an immune response for antibody production. In this work, we report the effect of multiple hapten loading on carrier protein after conjugation by monitoring the structural and immunogenic properties of the protein. Biochemical conjugation of carboxylated hapten (atrazine derivative) to bovine serum albumin via epsilon-amino groups of lysine residues was monitored by the intrinsic fluorescence intensity of tryptophan residues of protein. A significant blue shift of emission maxima confirmed the conformational changes with increasing molar ratio of hapten:protein. Circular dichroism spectroscopy suggested a decreasing trend for alpha-helical and increased formation of beta-sheet structures in hapten-loaded protein. A further insight was sought by using molecular modeling methods for understanding of structural changes in the native protein post-hapten conjugation. A sequential approach for hapten loading on the carrier confirmed that initial binding could affect the possible binding sites for subsequent incorporation of hapten molecules. These changes play a major role in the immunogenic response of hapten-carrier conjugate. The approach taken to develop this model is promising, and can be generalized for studies with other protein-hapten combinations.

Integrative proteogenomic analyses of human tumours identifies ADNP as a novel oncogenic mediator of cell cycle progression in high-grade serous ovarian cancer with poor prognosis.

BACKGROUND: Despite toxic side effects and limited durable response, the current standard-of-care treatment for high grade serous ovarian cancer (HGSOC) remains platinum/taxane-based chemotherapy. Given that the overall prognosis has not improved drastically over the past several decades, there is a critical need to understand the underlying mechanisms that lead to tumour development and progression. METHODS: We utilized an integrative proteogenomic analysis of HGSOC tumours applying a poor prognosis gene expression signature (PPS) as a conceptual framework to analyse orthogonal genomic and proteomic data from the TCGA (n = 488) and CPTAC (n = 169) studies. Genes identified through in silico analyses were assessed in vitro studies to demonstrate their impact on proliferation and cell cycle progression. FINDINGS: These analyses identified DNA amplification and overexpression of the transcription factor ADNP (Activity Dependent Neuroprotector Homeobox) in poorly prognostic tumours. Validation studies confirmed the prognostic capacity of ADNP and suggested an oncogenic role for this protein given the association between ADNP expression and pro-proliferative signalling. In vitro studies confirmed ADNP as a novel and essential mediator of cell proliferation through dysregulation of cell cycle checkpoints. INTERPRETATION: We identified ADNP as being amplified and overexpressed in poor prognosis HGSOC in silico analyses and demonstrated that ADNP is a novel and essential oncogene in HGSOC which mediates proliferation through dysregulation of cell cycle checkpoints in vitro. FUNDING: The National Cancer Institute of the National Institutes of Health, the V Foundation for Cancer Research and the New Jersey Commission for Cancer Research.

Behavioral alterations in rotenone model of Parkinson's disease: attenuation by co-treatment of centrophenoxine.

Rotenone, a potent specific inhibitor of mitochondrial complex-1, appears to reproduce the behavioral features of Parkinson's disease in rats. It destroys dopaminergic neurons selectively, causing deficiency of dopamine in striatum which leads to impaired motor functions. Oxidative stress generated as a result of mitochondrial dysfunction and metabolism of dopamine has been implicated as an important factor in the etiology of Parkinson's disease. Present study explores the potential of centrophenoxine (a well known anti-aging and antioxidant drug) against rotenone induced motor dysfunction. Sprague Dawley male rats were administered with rotenone on a daily basis by subcutaneous injection of dose: 2 mg/kg body weight over a period of 35 days. Data showed impaired motor function, significant increase in catalepsy, decrease in locomotor activity and decrease in muscle activity. Dopamine content of rotenone treated animals was found to decrease significantly and lipid peroxidation was found to increase significantly in rotenone treated animals when compared with co-treated group. Co-treatment with centrophenoxine (100 mg/kg i.p. for 35 days) significantly attenuated the extent of motor dysfunction and changes in the level of dopamine and lipid peroxidation induced by rotenone toxicity. Thus, the present study provides evidence that centrophenoxine co-treatment attenuates rotenone induced motor dysfunction by virtue of its antioxidant action.

Hidden burden of osteoporosis in alpha-1 antitrypsin deficiency.

This is a reminder of a rare cause of osteoporosis that remains widely underdiagnosed and lacks specific evidence on its optimal management. We bring a case report of a patient presenting with erectile dysfunction and high testosterone level but also elevated sex-hormone binding globulin hence low free androgen index as well as evidence of organ specific hypogonadal side effects such as osteoporosis. A unifying diagnosis of alpha-1 antitrypsin deficiency (AATD) brought together his coexistent mild chronic obstructive pulmonary disease as well as a new finding of previously unrecognised liver disease. This case highlights the uncertainties over the mechanism of osteoporosis in AATD as well as the controversies over best way to manage it. The efficacy of testosterone replacement in managing osteoporosis in the context of AATD remains untested. National registries for AATD would be best placed at gathering further evidence in this area.

Transmembrane segments form tertiary hairpins in the folding vestibule of the ribosome.

Folding of membrane proteins begins in the ribosome as the peptide is elongated. During this process, the nascent peptide navigates along 100Å of tunnel from the peptidyltransferase center to the exit port. Proximal to the exit port is a "folding vestibule" that permits the nascent peptide to compact and explore conformational space for potential tertiary folding partners. The latter occurs for cytosolic subdomains but has not yet been shown for transmembrane segments. We now demonstrate, using an accessibility assay and an improved intramolecular crosslinking assay, that the helical transmembrane S3b-S4 hairpin ("paddle") of a voltage-gated potassium (Kv) channel, a critical region of the Kv voltage sensor, forms in the vestibule. S3-S4 hairpin interactions are detected at an early stage of Kv biogenesis. Moreover, this vestibule hairpin is consistent with a closed-state conformation of the Kv channel in the plasma membrane.

Peripartum cardiomyopathy.

Peripartum cardiomyopathy remains a rare but troubling complication of pregnancy. The cardiovascular stress of labour and delivery may lead to cardiac decompensation. These patients require special anaesthetic care during labour and delivery. Compensated patients may undergo vaginal delivery with appropriate monitoring. For those patients late in pregnancy with more significant haemodynamic compromise, consideration should be given to elective caesarean section with invasive haemodynamic monitoring of the mother. We report two antenatal cases of peripartum cardiomyopathy where a near miss situation was avoided by doing an emergency caesarean section.

Upregulation of the Tim-3/galectin-9 pathway of T cell exhaustion in chronic hepatitis B virus infection.

The S-type lectin galectin-9 binds to the negative regulatory molecule Tim-3 on T cells and induces their apoptotic deletion or functional inactivation. We investigated whether galectin-9/Tim-3 interactions contribute to the deletion and exhaustion of the antiviral T cell response in chronic hepatitis B virus infection (CHB). We found Tim-3 to be expressed on a higher percentage of CD4 and CD8 T cells from patients with CHB than healthy controls (p<0.0001) and to be enriched on activated T cells and those infiltrating the HBV-infected liver. Direct ex vivo examination of virus-specific CD8 T cells binding HLA-A2/peptide multimers revealed that Tim-3 was more highly upregulated on HBV-specific CD8 T cells than CMV-specific CD8 T cells or the global CD8 T cell population in patients with CHB (p<0.001) or than on HBV-specific CD8 after resolution of infection. T cells expressing Tim-3 had an impaired ability to produce IFN-γ and TNF-α upon recognition of HBV-peptides and were susceptible to galectin-9-triggered cell death in vitro. Galectin-9 was detectable at increased concentrations in the sera of patients with active CHB-related liver inflammation (p = 0.02) and was strongly expressed by Kupffer cells within the liver sinusoidal network. Tim-3 blockade resulted in enhanced expansion of HBV-specific CD8 T cells able to produce cytokines and mediate cytotoxicity in vitro. Blocking PD-1 in combination with Tim-3 enhanced the number of patients from whom functional antiviral responses could be recovered and/or the strength of responses, indicating that these co-inhibitory molecules play a non-redundant role in driving T cell exhaustion in CHB. Patients taking antivirals able to potently suppress HBV viraemia continued to express Tim-3 on their T cells and respond to Tim-3 blockade. In summary, both Tim-3 and galectin-9 are increased in CHB and may contribute to the inhibition and deletion of T cells as they infiltrate the HBV-infected liver.