AMPK & Metformin links T cell aging during Spaceflight with Cancer stem cells, HIV-1 latency, telomeres, oocyte activation, & Progeria

Goldsmith Content Providers: CDC/ C. Goldsmith, P. Feorino, E. L. Palmer, W. R. McManus [Public domain], via Wikimedia Commons; The Cell Nucleus and Aging: Tantalizing Clues and Hopeful Promises. Scaffidi P, Gordon L, Misteli T. PLoS Biology Vol. 3/11/2005, e395; CC BY 2.5 (http://creativecommons.org/licenses/by/2.5)], via Wikimedia Commons; By NASA [Public domain], via Wikimedia Commons

A recent study published in the journal Oncogene in June of 2017 by researchers from The Johns Hopkins University School of Medicine and Emory University School of Medicine demonstrated that a bioactive molecule derived from Magnolia grandiflora significantly inhibited breast cancer stem cells (also known as tumor-initiating cells) in vivo in mice and also inhibited the expression of the stem cell/pluripotency markers Oct4, Nanog, and Sox2 in breast cancer cells in an AMPK-dependent manner [1]. Interestingly, a recent study published in the journal Oncotarget in May of 2017 showed that metformin activated AMPK and inhibited cell proliferation, clonogenic ability, tumorsphere formation (a characteristic of cancer stem cells), and the cancer stem cell population in chemotherapy-resistant colorectal cancer cells [2]. Metformin has also been shown to promote the differentiation and/or apoptosis of cancer stem cells in vitro and in vivo in an AMPK-dependent manner from the deadliest of cancers, including glioblastoma and pancreatic cancer [3]. Cancer stem cells (CSCs) exhibit several stem cell-like qualities that are characteristic of embryonic or adult stem cells, including self-renewal, differentiation, and the ability to initiate tumorigenesis. CSCs, similar to adult stem cells (ASCs), may also undergo quiescence, a state or period of inactivity or dormancy that contributes to the resistance of CSCs to radiation and/or chemotherapy, potentially allowing CSCs to re-seed tumors at a later time [3]. Intriguingly, HIV-1 has been shown to establish durable and long-stating latency, similar to CSC quiescence, in central CD4+ memory T cells as well as in T memory stem cells, T cell subsets that also display stem-cell like qualities including self renewal and differentiation. Latency establishment by HIV-1 in these T cell subsets prevents immune system detection and virus destruction or virus-induced cell death, analogous to CSC quiescence [3]. Because the use of AMPK activators, including metformin, bryostain-1, JQ1, and resveratrol as single agents or in a combinatorial fashion reactivates latent HIV-1 and induces CSC differentiation and/or apoptosis, a novel observation would logically follow that AMPK activation links the reactivation of latent HIV-1 (known as the “shock and kill” approach in HIV-1 cure research) with the “activation” (i.e. cell-cycle reentry), differentiation, and/or apoptosis of CSCs (which may also be labeled as a “shock and kill” approach) [3].

Indeed, a recently published paper in June of 2017 that I authored (and an additional paper currently under review) elucidates a novel link that connects CSCs with HIV-1 latency, microgravity and spaceflight, Hutchinson-Gilford progeria syndrome (HGPS), oocyte activation, the sperm acrosome reaction, Down syndrome, and hippocampal long-term potentiation [3,4]. This connection is predicated on the induction of cellular stress (mediated by increases in intracellular calcium [Ca2+] levels, reactive oxygen species [ROS] generation, and/or AMP/ATP ratio increases, etc), leading to beneficial cellular responses that are mediated by the master metabolic regulator AMPK [3]. As further explained below, AMPK is activated by a variety of stressors (osmotic, electrical stimulation, chemical, heat stress, etc.) as well as stressors that are diminished in microgravity/spaceflight (e.g. shear stress, force). AMPK also increases lifespan and healthspan in several model organisms (e.g. yeast, worms, flies, and mice) and metformin alleviates accelerated aging defects in cells from children with the genetic disorder HGPS. Because AMPK is also critical for T cell activation and the mounting of an effective immune response, promotes embryonic and adult stem cell differentiation, and metformin enhances bone formation, osteoprotegerin synthesis, and telomere integrity, AMPK activation by structurally diverse compounds that have a proven safety record may indeed provide protection for astronauts embarking on long-term space missions [3,5-7].

In May of 2017, I published for the first time that cellular stress-induced AMPK activation links the differentiation and/or apoptosis of cancer stem cells (CSCs) with the reactivation of latent HIV-1 and alleviation of accelerated aging-like cellular defects resulting from microgravity and spaceflight [3]. ROS and Ca2+ are well-studied mediators of cellular stress-induced differentiation of embryonic and adult stem cells, AMPK has recently been shown to be essential for mouse embryonic stem cell differentiation, and metformin targets and promotes differentiation and/or apoptosis of cancer stem cells in the deadliest of cancers in an AMPK-dependent manner, including glioblastoma and pancreatic cancer [3]. Interestingly, AMPK is critical for T cell activation and the mounting of an effective immune response in vivo, as AMPK inhibition during T cell activation leads to T cell death [3]. HIV-1 has been shown to establish latency (thus preventing viral eradication) in long-lived T cell subsets that display longer telomeres, including central CD4+ memory T cells and CD4+ T memory stem cells [3]. However, the phorbol ester PMA combined with the calcium ionophore ionomycin is used as a positive control to reactivate latent HIV-1 and both compounds have been shown in independent studies to activate AMPK. Several structurally diverse compounds that also induce reactivation of latent HIV-1 in CD4+ memory T cells (called the “shock and kill” approach in HIV-1 cure research), including metformin combined with bryostatin-1, butyrate, JQ1, etc. also promote the differentiation and/or apoptosis of CSCs, indicating that the cellular states of CSC quiescence and HIV-1 latency are analogous and the induction of a cellular stress response, mediated by AMPK, may lead to both CSC elimination and viral eradication [3].

Additionally, simulated microgravity has been shown to inhibit AMPK activation, transient levels of hypergravity activate AMPK, and shear stress and the application of force have both been shown to activate AMPK, indicating that gravity itself represents a cellular stressor and is essential for efficient T cell activation and the mounting of an effective immune response [3,8,9]. Indeed, the application of force enhances T cell activation and both embryonic stem cell differentiation and T cell activation are significantly inhibited during actual spaceflight [3]. The lack of cellular stress induction (i.e. mechano-transduction) in microgravity/spaceflight may also explain the recent unusual observance of longer telomeres in the flight-based compared to the ground-based subject in the NASA TWINS Study [10]. Central memory T cells and T memory stem cells (TSCMs) are long-lived T cell subsets with longer telomeres and are capable of self-renewal and differentiation into more differentiated effector T cells [11,12]. However, as noted above, cellular stress and subsequent AMPK activation is essential for T cell activation and the mounting of an effective immune response and microgravity/spaceflight minimizes cellular stress induction, leading to an increase in undifferentiated T cell subsets that possess stem-cell like qualities (e.g. longer telomeres), with a consequent deficiency in the response to viruses, bacteria, and pathogens that return to normal levels after re-introduction of cellular stress (i.e. return to Earth’s gravitational field) [3].  

Strikingly, cells that are placed in NASA-developed rotating wall vessel bioreactors (RWVs), devices that are commonly used to mimic microgravity that also reduce shear stress, exhibit stem cell-like behavior (i.e. sphere formation) and reduced differentiation, very much similar to cancer stem cells [13]. A recent study has also shown that multipotent cells treated with strontium chloride, a compound that activates mouse oocytes and has been used to activate human oocytes, producing normal healthy children, induced activation of osteoblastogenesis in RWVs, indicating that cellular stress, mediated by gravity/force (i.e. mechano-transduction), Ca2+, ROS, and/or AMP/ATP ratio increases, etc. links AMPK activation with microgravity/spaceflight, oocyte activation, cancer stem cells, and HGPS (see below) [14-16].

In 2014, I was the first to propose and publish that metformin would alleviate accelerated aging defects in cells derived from patients with the accelerated aging disorder HGPS by promoting cellular stress-induced AMPK activation and beneficially altering the activity of the gene splicing factor SRSF1 (which is dysregulated in HGPS) [17]. This hypothesis was substantiated in 2016 and 2017, with metformin activating AMPK, decreasing the levels of SRSF1, and alleviating accelerated aging defects in HGPS cells [18,19]. In 2015, I was also the first to publish that the gene splicing factor SRSF1 links HGPS and HIV-1 latency, as excessive splicing activity of SRSF1 prevents reactivation of HIV-1, preventing viral cytopathic or cytolytic effects [20]. Because AMPK activation is critical for T cell activation, I also hypothesized that structurally dissimilar compounds that induce latent HIV-1 reactivation actually do so via indirect cellular stress-induced AMPK activation [20]. Intriguingly, a study by researchers at Merck showed that knockdown of AMPK or CaMKK2 (an upstream kinase/activator of AMPK) significantly inhibits HIV-1 replication [21]. Several compounds and methodologies that reactivate latent HIV-1, including heat stress, free-radical generating compounds, resveratrol, and many others have been shown to induce AMPK activation, indicating that cellular stress-induced AMPK activation orchestrates and connects both latent HIV-1 reactivation and HGPS [3,20,25]. Interestingly, temsirolimus, a rapamycin analog, was recently shown to correct accelerated aging defects in HGPS cells but transiently increase the levels of ROS and superoxide anion but decrease VO2 max within the first hour, providing further evidence that cellular stress-induced AMPK activation links HIV-1 latency and HGPS [22]. Intriguingly, rapamycin production by the bacterium Streptomyces hygroscopicus is significantly inhibited (~90%) when the bacterium is exposed to conditions of microgravity (i.e. being placed in a rotating wall vessel bioreactor), indicating that the beneficial effects of cellular stress induction may cross species boundaries, leading to increased production of valuable secondary metabolites by microorganisms (e.g. penicillin, rapamycin, avermectins, etc.) [23].

In 2016, I also first proposed that AMPK activation links oocyte activation, which is a prerequisite for the creation of all human life, with the reactivation of latent HIV-1 reservoirs [24]. The intracellular signaling mechanisms that characterize oocyte activation are incredibly similar to those that characterize latent HIV-1 reactivation in memory CD4+ T cells, and include the PLC-PIP2-IP3-Ca2+ and PLC-PIP2-DAG-PKC pathways. AMPK activation is also critical for oocyte meiotic resumption and maturation, two processes that precede and are critical for efficient oocyte activation [24]. Also, PMA and ionomycin, both of which activate AMPK and are used as positive controls in HIV-1 latency reversal studies, also induce mouse oocyte activation (models for human oocytes) and ionomycin is extensively used during in vitro fertilization procedures to activate human oocytes, producing normal healthy children [24]. Interestingly, MG132 (proteasome inhibitor), methylene blue, menadione (free radical-generating agent), resveratrol analogs, heat stress, and osmotic stress have each been shown to induce mouse oocyte meiotic resumption and activate AMPK [24]. Additionally, AMPK has recently been found localized across the entire acrosome in human spermatozoa and stress-inducing compounds, including ROS, PMA, ionomycin, vitamin D, and capsaicin have each been shown to induce the acrosome reaction in human sperm, an indispensable process for the creation of all human life outside of a clinical setting [3,4,26]. Interestingly, electrical stimulation, puromycin (a protein synthesis inhibitor), and ethanol have also been shown to activate human oocytes and induce AMPK activation, suggesting that cellular stress-induced AMPK activation, mediated by Ca2+, ROS, and AMP/ATP ratio increases, etc. links the creation of all human life (i.e. the “shock and live” approach) with HGPS and latent HIV-1 reactivation (i.e. the “shock and kill” approach) [24].

Also, I have a recently submitted manuscript (2017) currently under review that links cellular stress-induced AMPK activation with hippocampal CA1 long-term potentiation (LTP, a process critical for learning and memory) and HIV-1 latency [4]. Indeed, an increase in intracellular Ca2+ levels is indispensable for the induction of hippocampal CA1 LTP, the inhibition of ROS production or ROS scavengers significantly inhibits hippocampal CA1 LTP, and the primary excitatory neurotransmitter glutamate activates AMPK in neurons and also enhances T cell activation/proliferation [4]. Metformin also activates AMPK in hippocampal CA1 neurons, resveratrol upregulates AMPA receptors in the hippocampus in an AMPK-dependent manner (AMPA receptors are considered critical for the expression of LTP), the phorbol ester PMA enhances hippocampal CA1 LTP, and metformin alleviates Aβ-mediated reductions in hippocampal LTP, indicating that cellular stress-induced AMPK activation links hippocampal CA1 LTP with HIV-1 latency, HGPS, cancer stem cells, oocyte activation, sperm acrosome reaction, and spaceflight [4]. Interestingly, metformin also improves behavioral defects and activates AMPK in a mouse model of Fragile X syndrome, reverses mitochondrial defects in human fibroblasts derived from fetuses with Down syndrome, and inhibition of the Down syndrome gene DYRK1A induces reactivation of latent HIV-1, indicating that cellular stress-induced AMPK activation is also beneficial for and links certain genetic disorders with HIV-1 latency [3,27].

In conclusion, accumulating evidence strongly suggests that the induction of cellular stress, mediated by increases in ROS, intracellular Ca2+, and/or AMP/ATP ratio increases, etc. leads to beneficial cellular responses that are orchestrated by the master metabolic regulator AMPK. As first proposed in my current and forthcoming publications, cellular stress-induced AMPK activation links the therapeutic effects of structurally diverse compounds in seemingly disparate physiological and patho-physiological states, including cancer stem cells, HIV-1 latency, microgravity and spaceflight, Hutchinson-Gilford progeria syndrome, oocyte activation, the sperm acrosome reaction, Down syndrome, Fragile X syndrome, and hippocampal long-term potentiation. As evidence continues to accrue substantiating this novel link, a paradigm shift in the assessment of disease etiology and the practice of medicine is inevitable.

https://www.linkedin.com/pulse/ampk-metformin-links-cell-aging-during-spaceflight-cancer-finley?published=t

 

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