The adenosinergic machinery in cancer: In-tandem insights from basic mechanisms to therapy.

Extracellular adenosine (eADO) signaling has emerged as an increasingly important regulator of immune responses, including tumor immunity. eADO is mainly produced from extracellular ATP (eATP) hydrolysis. eATP is rapidly accumulated in the extracellular space following cell death or cellular stress triggered by hypoxia, nutrient starvation, or inflammation. eATP plays a pro-inflammatory role by binding and activating the P2 purinergic receptors (P2X and P2Y), while eADO has been reported in many studies to mediate immunosuppression by activating the P1 purinergic receptors (A1, A2A, A2B, and A3) in diverse immune cells. Consequently, the hydrolysis of eATP to eADO alters the immunosurveillance in the tumor microenvironment (TME) not only by reducing eATP levels but also by enhancing adenosine receptor signaling. The effects of both P1 and P2 purinergic receptors are not restricted to immune cells. Here we review the most up-to-date understanding of the tumor adenosinergic system in all cell types, including immune cells, tumor cells, and stromal cells in TME. The potential novel directions of future adenosinergic therapies in immuno-oncology will be discussed.

TMEM176B Regulates AKT/mTOR Signaling and Tumor Growth in Triple-Negative Breast Cancer.

TMEM176B is a member of the membrane spanning 4-domains (MS4) family of transmembrane proteins, and a putative ion channel that is expressed in immune cells and certain cancers. We aimed to understand the role of TMEM176B in cancer cell signaling, gene expression, cell proliferation, and migration in vitro, as well as tumor growth in vivo. We generated breast cancer cell lines with overexpressed and silenced TMEM176B, and a therapeutic antibody targeting TMEM176B. Proliferation and migration assays were performed in vitro, and tumor growth was evaluated in vivo. We performed gene expression and Western blot analyses to identify the most differentially regulated genes and signaling pathways in cells with TMEM176B overexpression and silencing. Silencing TMEM176B or inhibiting it with a therapeutic antibody impaired cell proliferation, while overexpression increased proliferation in vitro. Syngeneic and xenograft tumor studies revealed the attenuated growth of tumors with TMEM176B gene silencing compared with controls. We found that the AKT/mTOR signaling pathway was activated or repressed in cells overexpressing or silenced for TMEM176B, respectively. Overall, our results suggest that TMEM176B expression in breast cancer cells regulates key signaling pathways and genes that contribute to cancer cell growth and progression, and is a potential target for therapeutic antibodies.

The midlife transition and the risk of cardiovascular disease and cancer Part I: magnitude and mechanisms.

Heart disease and cancer are the leading causes of death in the United States. In women, the clinical appearance of both entities-coronary heart disease and cancer (breast, endometrium, and ovary)-escalate during the decades of the midlife transition encompassing the menopause. In addition to the impact of aging, during the interval between the age of 40 and 65 years, the pathophysiologic components of metabolic syndrome also emerge and accelerate. These include visceral adiposity (measured as waist circumference), hypertension, diabetes, and dyslipidemia. Osteoporosis, osteoarthritis, sarcopenia, depression, and even cognitive decline and dementia appear, and most, if not all, are considered functionally related. Two clinical reports confirm the interaction linking the emergence of disease: endometrial cancer and metabolic syndrome. One describes the discovery of unsuspected endometrial cancer in a large series of elective hysterectomies performed in aged and metabolically susceptible populations. The other is from the Women's Health Initiative Observational Study, which found a positive interaction between endometrial cancer and metabolic syndrome regardless of the presence or absence of visceral adiposity. Both provide additional statistical support for the long-suspected causal interaction among the parallel but variable occurrence of these common entities-visceral obesity, heart disease, diabetes, cancer, and the prevalence of metabolic syndrome. Therefore, 2 critical clinical questions require analysis and answers: 1: Why do chronic diseases of adulthood-metabolic, cardiovascular, endocrine-and, in women, cancers of the breast and endometrium (tissues and tumors replete with estrogen receptors) emerge and their incidence trajectories accelerate during the postmenopausal period when little or no endogenous estradiol is available, and yet the therapeutic application of estrogen stimulates their appearance? 2: To what extent should identification of these etiologic driving forces require modification of the gynecologist's responsibilities in the care of our patients in the postreproductive decades of the female life cycle? Part l of this 2-part set of "expert reviews" defines the dimensions, gravity, and interactive synergy of each clinical challenge gynecologists face while caring for their midlife (primarily postmenopausal) patients. It describes the clinically identifiable, potentially treatable, pathogenic mechanisms driving these threats to quality of life and longevity. Part 2 (accepted, American Journal of Obstetrics & Gynecology) identifies 7 objectives of successful clinical care, offers "triage" prioritization targets, and provides feasible opportunities for insertion of primary preventive care initiatives. To implement these goals, a reprogrammed, repurposed office visit is described.

Gαq Sensitizes TRPM8 to Inhibition by PI(4,5)P2 Depletion upon Receptor Activation.

The cold- and menthol-sensitive transient receptor potential melastatin 8 (TRPM8) channel is important for both physiological temperature detection and cold allodynia. Activation of G-protein-coupled receptors (GPCRs) by proinflammatory mediators inhibits these channels. It was proposed that this inhibition proceeds via direct binding of Gαq to the channel. TRPM8 requires the plasma membrane phospholipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2 or PIP2] for activity. However, it was claimed that a decrease in cellular levels of this lipid upon receptor activation does not contribute to channel inhibition. Here, we show that supplementing the whole-cell patch pipette with PI(4,5)P2 reduced inhibition of TRPM8 by activation of Gαq-coupled receptors in mouse dorsal root ganglion (DRG) neurons isolated from both sexes. Stimulating the same receptors activated phospholipase C (PLC) and decreased plasma membrane PI(4,5)P2 levels in these neurons. PI(4,5)P2 also reduced inhibition of TRPM8 by activation of heterologously expressed muscarinic M1 receptors. Coexpression of a constitutively active Gαq protein that does not couple to PLC inhibited TRPM8 activity, and in cells expressing this protein, decreasing PI(4,5)P2 levels using a voltage-sensitive 5'-phosphatase induced a stronger inhibition of TRPM8 activity than in control cells. Our data indicate that, upon GPCR activation, Gαq binding reduces the apparent affinity of TRPM8 for PI(4,5)P2 and thus sensitizes the channel to inhibition induced by decreasing PI(4,5)P2 levels.SIGNIFICANCE STATEMENT Increased sensitivity to heat in inflammation is partially mediated by inhibition of the cold- and menthol-sensitive transient receptor potential melastatin 8 (TRPM8) ion channels. Most inflammatory mediators act via G-protein-coupled receptors that activate the phospholipase C pathway, leading to the hydrolysis of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. How receptor activation by inflammatory mediators leads to TRPM8 inhibition is not well understood. Here, we propose that direct binding of Gαq both reduces TRPM8 activity and sensitizes the channel to inhibition by decreased levels of its cofactor, PI(4,5)P2 Our data demonstrate the convergence of two downstream effectors of receptor activation, Gαq and PI(4,5)P2 hydrolysis, in the regulation of TRPM8.

Diabetes, Obesity, and Breast Cancer.

The rates of obesity and diabetes are increasing worldwide, whereas the age of onset for both obesity and diabetes are decreasing steadily. Obesity and diabetes are associated with multiple factors that contribute to the increased risk of a number of different cancers, including breast cancer. These factors are hyperinsulinemia, elevated IGFs, hyperglycemia, dyslipidemia, adipokines, inflammatory cytokines, and the gut microbiome. In this review, we discuss the current understanding of the complex signaling pathways underlying these multiple factors involved in the obesity/diabetes-breast cancer link, with a focus particularly on the roles of the insulin/IGF system and dyslipidemia in preclinical breast cancer models. We review some of the therapeutic strategies to target these metabolic derangements in cancer. Future research directions and potential therapeutic strategies are also discussed.

Normalization of connexin 43 protein levels prevents cellular and functional signs of dystrophic cardiomyopathy in mice.

Duchenne muscular dystrophy (DMD) associated cardiomyopathy remains incurable. Connexin 43 (Cx43) is upregulated and remodeled in the hearts of mdx mice, a mouse model of DMD. Hearts from Wild Type, mdx, and mdx:Cx43(+/-) mice were studied before (4-6 months) and after (10-15 months) the onset of cardiomyopathy to assess the impact of decreasing Cx43 levels on cardiac pathology in dystrophic mice. Increased connexin 43 protein levels in mdx hearts were not observed in mdx:Cx43(+/-) hearts. Cx43 remodeling in mdx hearts was attenuated in mdx:Cx43(+/-) hearts. At time-point 4-6 months, isolated cardiomyocytes from mdx hearts displayed enhanced ethidium bromide uptake, augmented intracellular calcium signals and increased production of reactive oxygen species. These pathological features were improved in mdx:Cx43(+/-) cardiomyocytes. Isoproterenol-challenged mdx:Cx43(+/-) mice did not show arrhythmias or acute lethality observed in mdx mice. Likewise, isoproterenol-challenged mdx:Cx43(+/-) isolated hearts were also protected from arrhythmogenesis. At time-point 10-15 months, mdx:Cx43(+/-) mice showed decreased cardiac fibrosis and improved ventricular function, relative to mdx mice. These results suggest that normalization of connexin 43 protein levels in mdx mice reduces overall cardiac pathology.

Deficit in PINK1/PARKIN-mediated mitochondrial autophagy at late stages of dystrophic cardiomyopathy.

Aims: Duchenne muscular dystrophy (DMD) is an inherited devastating muscle disease with severe and often lethal cardiac complications. Emerging evidence suggests that the evolution of the pathology in DMD is accompanied by the accumulation of mitochondria with defective structure and function. Here, we investigate whether defects in the housekeeping autophagic pathway contribute to mitochondrial and metabolic dysfunctions in dystrophic cardiomyopathy. Methods and results: We employed various biochemical and imaging techniques to assess mitochondrial structure and function as well as to evaluate autophagy, and specific mitochondrial autophagy (mitophagy), in hearts of mdx mice, an animal model of DMD. Our results indicate substantial structural damage of mitochondria and a significant decrease in ATP production in hearts of mdx animals, which developed cardiomyopathy. In these hearts, we also detected enhanced autophagy but paradoxically, mitophagy appeared to be suppressed. In addition, we found decreased levels of several proteins involved in the PINK1/PARKIN mitophagy pathway as well as an insignificant amount of PARKIN protein phosphorylation at the S65 residue upon induction of mitophagy. Conclusions: Our results suggest faulty mitophagy in dystrophic hearts due to defects in the PINK1/PARKIN pathway.

Oxidative stress and ca(2+) release events in mouse cardiomyocytes.

Cellular oxidative stress, associated with a variety of common cardiac diseases, is well recognized to affect the function of several key proteins involved in Ca(2+) signaling and excitation-contraction coupling, which are known to be exquisitely sensitive to reactive oxygen species. These include the Ca(2+) release channels of the sarcoplasmic reticulum (ryanodine receptors or RyR2s) and the Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). Oxidation of RyR2s was found to increase the open probability of the channel, whereas CaMKII can be activated independent of Ca(2+) through oxidation. Here, we investigated how oxidative stress affects RyR2 function and SR Ca(2+) signaling in situ, by analyzing Ca(2+) sparks in permeabilized mouse cardiomyocytes under a broad range of oxidative conditions. The results show that with increasing oxidative stress Ca(2+) spark duration is prolonged. In addition, long and very long-lasting (up to hundreds of milliseconds) localized Ca(2+) release events started to appear, eventually leading to sarcoplasmic reticulum (SR) Ca(2+) depletion. These changes of release duration could be prevented by the CaMKII inhibitor KN93 and did not occur in mice lacking the CaMKII-specific S2814 phosphorylation site on RyR2. The appearance of long-lasting Ca(2+) release events was paralleled by an increase of RyR2 oxidation, but also by RyR-S2814 phosphorylation, and by CaMKII oxidation. Our results suggest that in a strongly oxidative environment oxidation-dependent activation of CaMKII leads to RyR2 phosphorylation and thereby contributes to the massive prolongation of SR Ca(2+) release events.

Hierarchical accumulation of RyR post-translational modifications drives disease progression in dystrophic cardiomyopathy.

AIMS: Duchenne muscular dystrophy (DMD) is a muscle disease with serious cardiac complications. Changes in Ca(2+) homeostasis and oxidative stress were recently associated with cardiac deterioration, but the cellular pathophysiological mechanisms remain elusive. We investigated whether the activity of ryanodine receptor (RyR) Ca(2+) release channels is affected, whether changes in function are cause or consequence and which post-translational modifications drive disease progression. METHODS AND RESULTS: Electrophysiological, imaging, and biochemical techniques were used to study RyRs in cardiomyocytes from mdx mice, an animal model of DMD. Young mdx mice show no changes in cardiac performance, but do so after ∼8 months. Nevertheless, myocytes from mdx pups exhibited exaggerated Ca(2+) responses to mechanical stress and 'hypersensitive' excitation-contraction coupling, hallmarks of increased RyR Ca(2+) sensitivity. Both were normalized by antioxidants, inhibitors of NAD(P)H oxidase and CaMKII, but not by NO synthases and PKA antagonists. Sarcoplasmic reticulum Ca(2+) load and leak were unchanged in young mdx mice. However, by the age of 4-5 months and in senescence, leak was increased and load was reduced, indicating disease progression. By this age, all pharmacological interventions listed above normalized Ca(2+) signals and corrected changes in ECC, Ca(2+) load, and leak. CONCLUSION: Our findings suggest that increased RyR Ca(2+) sensitivity precedes and presumably drives the progression of dystrophic cardiomyopathy, with oxidative stress initiating its development. RyR oxidation followed by phosphorylation, first by CaMKII and later by PKA, synergistically contributes to cardiac deterioration.

Mutation of the conserved Asp122 in the linker impedes creatine kinase reactivation and refolding.

Creatine kinase (CK), a key enzyme in maintaining the intracellular energetic homeostasis, contains two domains connected by a long linker. In this research,we found that the mutations of the conserved Asp122 in the linker slightly affected CK activity, structure and stability. The hydrogen bonding and the ion pair contributed 2-5 kJ/mol to the conformational stability of CK. Interestingly, the ability of CK reactivation from the denatured state was completely removed by the mutations. These results suggested that the electrostatic interactions were crucial to the action of the linker in CK reactivation.