Commentary on Some Recent Theses Relevant to Combating Aging: April 2016

Cancer Exosomes Are Unique and Complex Mechanisms that Suppress Effector T-Lymphocyte Functions

Yueting Wu, PhD, West Virginia University

It is still unknown how tumor exosomes influence malignant cell survival and alter cell-to-cell communication to modulate the immune system by, in part, manipulating the activity of cytotoxic T lymphocytes. This study focuses on two critical parts regarding cancer exosomes. The first part is to tailor existing bionanotechnology methods to account for the nanoscale aspects of exosome biology. SEM, TEM, Bioanalyzer, and flow cytometry were used to characterize exosome morphologies, identify specific protein biomarkers HER1 and HER2, as well as the quality of RNAs enclosed in exosomes. Competing methods related to exosome isolation, production, preservation, stability, and analysis were evaluated. Based on these studies, we recommend improved experimental methods that aim to ensure a consistent framework to identify the roles that exosomes play. With these improved methods, the second part is to characterize the immunosuppressive role that melanoma exosomes play, especially from the perspective of delivering a payload of mRNAs to immune cells. Toward this second aim, melanoma exosomes were purified and cytokine receptor IL12Rβ2 and specific mRNA enrichment were identified. Microarray and pathway analysis suggested that mRNAs derived from melanoma impact a variety of immune signaling pathways. Induction effects of PTPN11 and DNMT3A from the exosomal mRNAs were characterized in T lymphocytes. Specifically, we showed that PTPN11 upregulation impeded CTLL-2 cytotoxic T-cell proliferation in response to IL2 stimulation, and DNMT3A upregulation hindered IFN-γ production in 2D6 TH1 cells. These findings provide insights regarding the specific immunosuppression effects that tumor-infiltrating lymphocytes may encounter in tumor microenvironment. Understanding those immunosuppression effects is important to engineer antitumor immunity for innovative and improved treatments against cancer.

Comment: Exosomes are tiny membrane-bound vesicles secreted by cells into the surrounding medium and are now thought to be present in most, if not all, biological fluids. Derived from the endosomal network and released either by direct budding from the plasma membrane, or shed when multivesicular bodies fuse with it, exosomes incorporate a distinctive complement of conserved proteins as well as cell type-specific proteins and RNA. Although they are remarkably stable in vivo, their isolation and analysis have proven challenging; at 30–100 nm in diameter, they are difficult to separate from lipoprotein particles by centrifugation and too small to detect by conventional optical microscopy. The initial focus of this dissertation on methodological standardization is thus highly commendable, and indeed a crucial contribution to any rapidly growing field. Despite the technical obstacles, it is now well established that exosomes play an important role in intercellular communication in both healthy and diseased states. A recent initiative to exploit their ability to cross the blood–brain barrier and deliver materials to neurons has received considerable attention (and funding), but their significance in the context of cancer and immunity continues to be the most active area of research. Cancer-derived exosomes have been shown to be involved in reprogramming stromal cells to a tumor-supportive or actively neoplastic state (a role for which they are particularly well suited given their ability to deliver transcriptionally active RNA, which can suffice to promote tumorigenesis ¹² ), and this combined with their ready dissemination throughout the body has fostered a growing suspicion that they may be critical facilitators of metastasis. However, their function in suppressing immune attack may be just as pathologically relevant; the natural role of exosomes in coordination between antigen-presenting cells and T cells renders them an obvious avenue for cancers to coopt to defuse the latter. While immunotherapy is swiftly proving to be a potent weapon in the oncologist's arsenal, established cancers evade immune attack through multiple parallel mechanisms, and the mixed results observed in some trials are surely attributable, in part, to variation in the susceptibility of the introduced cells to the defences of each particular tumor. Analyzing the exosomal complement secreted by a given cancer will add a new dimension to our understanding of those defences and drive the development of more reliably effective immunotherapies. Of course, should it be discovered that the uptake of exosomes is a wholly active process–currently quite uncertain–then it may be possible to engineer therapeutic T cells to resist this particular type of subversion entirely.

Senescent Fibroblasts Drive Tumorigenesis Through the Establishment of an Immunosuppressive Microenvironment

Megan Ruhland, PhD, Washington University in St. Louis

The risk for developing cancer increases with age. This well-established phenomenon is likely driven, in part, by age-related increases in stochastic, epithelial genetic mutations. Cancer requires that numerous complementary mutations occur in a single incipient tumor cell. Consequently, it is not surprising that time is needed for mutations to accumulate and transformation to occur. In addition to changes within the epithelium, there is growing appreciation for the importance of the stromal compartment in tumorigenesis. In an established tumor microenvironment, cancer-associated fibroblasts can fuel tumor progression. In addition, activated stromal cells such as myofibroblasts and senescent fibroblasts can greatly influence both the transformation process in vitro and the progression of tumor development in vivo. Previous work has focused on the ability of tumors to coopt the microenvironment and thus drive progression. However, whether protumorigenic stromal changes can occur independent of neoplasia remains an unanswered question.

Senescent stromal cells accumulate in tissues with age and influence transformation and tumor progression. The mechanisms by which a senescent stromal compartment drives tumorigenesis remain to be fully elucidated. While senescent cells secrete growth factors that directly promote tumor growth, whether senescent cells can function in a nontransformed tissue to create a protumorigenic microenvironment remains largely unknown. Interestingly, senescent cells secrete elevated levels of cytokines and chemokines as part of their distinctive secretory profile. Whether the secretion of these factors impacts inflammation and tumor development in older individuals remains an important and unaddressed question.

The lack of immune-competent mouse models for investigating the specific impact of stromal senescence has made it difficult to interrogate the interplay between these cells and the immune response. My work directly investigates the consequences of stromal senescence on inflammation through the use of a novel mouse model. This unique model allows for both spatial and temporal control of the induction of senescence. Using this system, I identified a role for senescence in the establishment of an immunosuppressive microenvironment before the appearance of a neoplastic clone. I demonstrated that senescence-derived IL-6 drives immunosuppression by promoting myeloid-derived suppressor cell (MDSC) accumulation. In addition, I determined that senescence-mediated MDSC accumulation functions to promote tumor growth through CD8+ T-cell suppression. Furthermore, I showed that targeting MDSCs directly, or depleting senescence-secreted factor IL-6, could reverse the tumor-permissive changes in the stroma. My work highlights a previously unidentified role for senescent stromal cells in dictating protumorigenic immune responses and suggests that IL-6 may be a beneficial target for reducing immunosuppression in aged microenvironments.

Comment: The phenotype of cellular senescence has some benevolent physiological roles, notably in the context of wound healing. However, the steady accumulation of senescent cells with age has long been implicated in physiological decline, and the recent discovery of the proinflammatory senescence-associated secretory phenotype (SASP) has now provided an explanation for how the comparatively small population of such cells can exert significant systemic effects. Although SASP factors can readily be shown to enhance the growth of existing tumors, it was previously unclear whether the mere presence of senescent cells could create an environment permissive for an initial neoplastic transformation. This thesis employs the new FASST (Fibroblasts Accelerate Stromal-Supported Tumorigenesis) model–in which a tissue-specific Cre recombinase is employed to trigger the onset of p27^Kip1-induced senescence in a controlled manner–to provide the first definitive evidence that this is indeed the case, as well as elements of the mechanism involved. Work in other genetically engineered models, particularly those bearing the INK-ATTAC construct, has recently demonstrated an exceptionally robust reversal of multiple age-related maladies resulting from the ablation of senescent cells ¹³ ; translational efforts to develop small molecules (“senolytics”) capable of achieving the same result in unmodified organisms have accordingly enjoyed a dramatic increase in attention and funding. Treating cancer after it becomes established remains perhaps the greatest challenge of modern medicine, and the key implication of this work–that prophylactic use of senolytics should meaningfully reduce the age-related increase in tumor incidence–serves to further emphasize the importance of that line of research.

Targeting Lysosphingolipid-Induced Lysosomal Impairment as a Critical Point for Therapeutic Intervention in Lysosomal Storage Disorders

Nicole Scott, PhD, University of Rochester

Lysosomes play an important role in multiple cellular processes, including bacterial clearance, ion homeostasis, and nutrient sensing, in addition to their canonical role as a catabolic site for biomolecules and organelles. While lysosomal dysfunction is thought to be involved in multiple degenerative diseases, lysosomal storage disorders (LSDs) offer a unique pathology in which lysosomal impairment may be specifically addressed. LSDs comprised about 50 genetic disorders that result from defects in normal lysosomal function, most commonly through enzymatic deficiency, leading to an accumulation of undegraded substrates. While most therapies aim to restore the missing enzyme; through direct enzyme replacement, gene therapy, or cell transplant; our efforts have focused on understanding the underlying mechanisms of toxicity associated with substrate accumulation and how these mechanisms may affect the efficacy of these replacement therapies. We found that a group of toxic lipids that accumulate in several related LSDs, the lysosphingolipids, disrupt multiple cellular functions, specifically through the impairment of lysosomal activity caused by elevated lysosomal pH. Unbiased drug discovery revealed several protective compounds capable of preventing lysosomal and cellular toxicities caused by these lipids. Upon further evaluation, these compounds showed unexpected convergence on the control of lysosomal pH and reacidification as a critical component of protection. In addition to protective compounds, multiple compounds capable of enhancing lipid toxicity were also identified by our screen and appeared to again converge on lysosomal function. Many of the compounds identified were observed to impair the lysosomal activity independent of lipid accumulation, indicating that alterations in lysosomal function may be a critical component in the underlying sensitivity and vulnerability to substrate accumulation. Our studies thus reveal a novel hierarchy to substrate toxicity in several LSDs, wherein perturbations to lysosomal function mediated, at least in part, through changes in lysosomal pH, may not only contribute to disease progression but also may be a critical point of therapeutic intervention. Compounds that either ameliorate or exacerbate the underlying lysosomal dysfunction caused by substrate accumulation may be important clinically; potentially enhancing or antagonizing the efficacy of long-term treatment options aimed at restoring the enzymatic activity.

Comment: Despite their low incidence, collectively affecting only around 1 in 7500 newborns, congenital lysosomal disorders exhibit a devastating and progressive pathology that has led to intense study since the first such condition, Pompe disease, was identified in 1963. Most are characterized by the lysosomal accumulation of specific families of molecules, reflecting the absence or dysfunction of a key catabolic enzyme; a few arise instead from failures of lysosome biogenesis or trafficking, leading to a general backlog of waste material throughout the cell. The former, at least, have a comparatively obvious therapeutic option–restoring the missing enzymatic capacity, either transiently by direct infusion or more permanently through cell or gene therapy–and considerable progress has been made along exactly these lines, with over a dozen formulations in clinical use or currently undergoing phase 3 trials. It is now becoming clear that a similar, although much more slowly developing, decline in proper lysosomal function occurs in a number of age-related diseases, including one of the biggest killers–atherosclerosis–the progression of which is strongly associated with an accumulation of the oxidized cholesterol derivative 7-ketocholesterol in the lysosomes of vessel wall-resident macrophages. Unlike the congenital disorders, it is considered likely that the species driving age-related lysosomal disease are produced too slowly for an adequate catabolic mechanism to have evolved at all. Such conditions can potentially still be reversed by the introduction of novel lysosomal enzymes optimized to degrade the recalcitrant species; however, this approach relies on the assumption that the built-up waste has a causal role in pathogenesis–a premise which seems generally probable, but is not universally certain–and additionally depends on the identification or de novo design of a distinct therapeutic enzyme for each target. A more general method for bolstering the function of failing lysosomes would be greatly desirable; all the more so should it be effective in both congenital lysosomal disorders (a well-established indication, facilitating regulatory approval) and in age-related conditions (for which the same agent could subsequently have a far more general off-label use). Thus, we eagerly look forward to discovering whether the pH-restorative compounds identified in this thesis are also beneficial in lysosomal disorders unrelated to lysosphingolipid accumulation.

The Impact of Germline Genetic Variability on Chemotherapy-Induced Toxicity and Survival in Early Breast Cancer Patients

Christof Vulsteke, PhD, Universiteit Antwerpen (Belgium)

An important part of breast cancer research is focused on genetic biomarkers in which much of the focus has been on somatic mutations determined on tumor tissue (matching the treatment to the cancer).

However, it should not be forgotten that we also need to ensure the treatment is matched to the patient. In fact, cancer patients receiving chemotherapy display a large patient-to-patient variability, which is of major clinical importance, since it can lead to therapeutic failures or adverse drug reactions. Therefore, we wanted to investigate if germline genetic biomarkers impact on toxicity in early breast cancer patients allowing a better match of the treatment to the patient. We were interested in polymorphisms that were already reported to impact on toxicity/efficacy, as well as in polymorphisms involved in the metabolism of the studied drugs. However, the studies, which assessed the influence of single-nucleotide polymorphisms (SNPs) on toxicity/outcome, have been small in size or were limited to testing only a single or a few SNPs at most, thereby limiting the validity and applicability of the obtained results. The initial aim of our research was to assess a comprehensive and large set of relevant SNPs in key metabolic genes in a large study cohort receiving the same chemotherapy for early BC and study its correlation with development of short-term severe toxicity. In a second phase of our research, we wanted to assess the impact of these SNPs on outcome (relapse-free interval and breast cancer-specific survival). The third phase was to develop a predictive model of patient-related, chemotherapy-related, and genetic risk factors on hematological toxicity. During the final phase of our research, long-term toxicity was evaluated.

Our research evidently demonstrated that germline genetic variability, especially in the ATP binding cassette (ABC) genes encoding for the human ABC transporter family, has an impact on chemotherapy-induced toxicity and outcome of early breast cancer patients and can partially explain the large patient-to-patient variability observed. Given the fact that breast cancer is the first most frequent cancer in women and the leading cause of cancer-related deaths, further research in the field of pharmacogenomics is crucial.

Comment: The overall outcome from any medical intervention is a combination of beneficial on-target effects and undesired side effects. For much of history, while medical practice was aimed largely at infectious diseases, the latter part of the equation tended to be negligible; microbial biology differs sufficiently from ours that drugs targeting a particular pathogen typically had little interaction with our own cells. The shift in recent decades to a focus on chronic and age-related conditions, where there is often no clear distinction between patient and pathogen, has necessitated increasingly close attention to unintended consequences. Such consequences are of course not determined solely by the therapeutic agent; rather, they are the product of interactions between that agent and the recipient's unique biological makeup, so predicting their occurrence and magnitude is dependent on having as much data about the patient as possible. This thesis makes considerable headway in demonstrating the value of that approach, identifying correlations between particular genetic variants and the magnitude of chemotherapy-induced side effects. Yet, even a comprehensive panel of SNPs captures only a fraction of the data available. When one considers the sharply falling costs of genome sequencing (now around $1000) in contrast to the cost of conducting clinical trials (which frequently run to $40,000 per participant, and particularly in oncology can exceed $100,000), it is somewhat difficult to understand why it is not already customary to acquire whole-genome data to better inform the final analysis. We sincerely hope that this work and others like it will make the value of deeply understanding the patient population more apparent to trial coordinators.

The Pathophysiological Mechanisms of Human Amylin-Induced Stress in Pancreatic Cells

Sanghamitra Singh, DSc, The George Washington University

Human amylin (hA) is a small 37 amino acid peptide hormone secreted by pancreatic β-cells along with insulin in response to glucose stimulus. It misfolds to form insoluble aggregate human islet amyloid polypeptide, which has been shown to induce oxidative stress and toxicity in cells. While hA is amyloidogenic, rat amylin lacks this ability due to proline substitutions in IAPP. The pathology of type II diabetes mellitus is characterized by excessive extracellular and intracellular accumulation of toxic amylin species, soluble oligomers and insoluble fibrils in islets, eventually leading to β-cell loss. However, the exact mechanism of clearance of amylin and exactly how amyloid proteins, such as the pancreatic hormone amylin, aggregate and kill cells is still unclear. Hence, the main goal of this study has been to elucidate the molecular mechanisms and pathways involved in hA turnover and toxicity.

Elevated serum copper levels are often implicated in diabetics, but the significance of this event in conjunction with hA toxicity is still unclear. In addition, studies on another amyloid protein, brain β-amyloid, have shown that copper exposure exacerbates β-amyloid pathology. Therefore, studies outlined in the first chapter involved exploring the possibility of an interaction between amylin and copper and its subsequent effect on hA-induced cytotoxicity. The findings of this study suggested that hA forms a metalocomplex with Cu²⁺, which surprisingly mitigated metalocatalyzed ROS accumulation by quenching H₂O₂ and decreasing hydroxyl radical formation. In line with this finding, hA-Cu²⁺ complex also inhibited hA cytotoxicity by reversing mitochondrial dysfunction and preventing caspase and JNK activation, suggesting a novel and unexpected protective role of hA-Cu²⁺ complex in pancreatic cells.

While previous studies showed that hA is internalized by pancreatic cells and causes mitochondrial dysfunction, the precise cellular mechanisms and compartments involved in its turnover and degradation remain unclear. Hence, in the second chapter of this thesis, using confocal microscopy and biochemical approaches, the roles of the main cellular compartments and organelles such as mitochondria, nucleus, cytosol, Golgi apparatus, and lysosomes in hA clearance and detoxification were explored. In addition, the role of the main cellular proteolytic complex, the proteasome, was also explored. Results suggest that hA, following its internalization, first accumulates in the cytosol of pancreatic β-cells followed by its translocation into nucleus and, to a lesser extent, lysosomes. An increase in hA accumulation in the nucleus of pancreatic cells is proportional to its cytotoxicity. Cell fractionation, immunoprecipitation, and confocal microscopy studies revealed that hA interacts with the catalytic subunits of the proteasome in the nucleus, which results in a decrease in the 20S proteolytic activity, ubiquitination, and subsequent protein stress. Inhibition of proteasome activity in turn causes a significant increase in hA accumulation and toxicity, suggesting a pivotal role of the proteasomes in hA turnover and detoxification in pancreatic cells.

While others and we have shown that hA induces cytotoxicity in pancreatic cells by activation of stress kinases such as JNK and p38 leading to apoptosis, the upstream factors and signaling pathways regulating their activation remain undefined. In an effort to elucidate the molecular mechanism of hA toxicity in pancreatic cells, in the third chapter, the role of apoptotic signaling kinase, ASK1, in this process was investigated. ASK1 is known to be a common target of a wide range of stressors such as free radicals, toxic amyloid proteins, and possibly hA. Results revealed that in the course of its toxicity, human amylin also activates ASK1 suggesting its participation in hA evoked β-cell death. Indeed, inhibition of ASK1 activity subsequently attenuates hA cytotoxicity. This finding points to a redox-sensitive ASK1-mediated hA toxicity in pancreatic cells.

Comment: Type I diabetes is characterized by a dramatic loss of pancreatic β-cells in early life, resulting from autoimmune attack triggered by one or more of a still unclear range of initial stressors. Conversely, in type II diabetes, β-cell loss is a consequence, rather than a cause, of pathology, although a consequence that plays a major role in rendering the disease ultimately irreversible. The hallmark of type II diabetes is insulin resistance–typically due to dietary and lifestyle factors–which results in a compensatory increase in the production of proinsulin, and also of proIAPP (as the two share a common promoter). ProIAPP generation is stimulated further still in the context of systemic inflammation, and particularly of elevated TNFα, which at least partially underlies the age-related increase in the incidence of type II diabetes (along with a more general dysregulation of insulin pathway signalling ¹⁴ ). This excessive production overwhelms the cell's capacity for posttranslational N-terminal cleavage of proIAPP, leading to the accumulation of unprocessed precursor protein within secretory vesicles. In this study, it acts as a seed for the aggregation of mature IAPP into amyloid particles, a process that continues in the extracellular space once the contents of the vesicle are released from the cell. This dissertation advances in several ways our understanding of how such deposits impair β-cell function and viability following their reinternalization. The involvement of proteasome inhibition by amylin is of particular interest; proteotoxic stress is a common factor in many age-related conditions, and a general upregulation of protein recycling is likely to be a component of any comprehensive rejuvenation strategy, ¹⁵ as well as being a predictor of successful aging. ¹⁶ As in other amyloid-driven pathologies, the removal of existing aggregates by precisely targeted immunotherapy ¹⁷ will be critical in achieving this goal, especially in advanced cases of the disease.

The Role of Ribosome Biogenesis in Exercise-Induced Skeletal Muscle Anabolism in Aging

Michael Stec, PhD, The University of Alabama at Birmingham

Numerous chronic medical conditions, as well as normal aging, result in a significant loss of skeletal muscle mass. This has profound effects on quality of life and can increase the risk of all-cause mortality. Currently, the most potent treatment for reversing the loss of muscle mass is resistance exercise training (RT); however, the human muscle fiber growth (hypertrophy) response to this treatment is quite variable, and older adults do not respond as favorably to this treatment as younger adults. The focus of this dissertation is to elucidate the role that ribosome biogenesis plays in regulating the RT-induced hypertrophic response. We show that markers of ribosome biogenesis are attenuated in older adult muscle following an acute single bout of resistance exercise, indicating a possible mechanism regulating the age-induced blunted hypertrophic response to RT. In addition, using a K-means cluster analysis, we show that those older individuals with a successful hypertrophic response to a standardized RT program significantly increase muscle rRNA production, and this is associated with a marked induction of c-Myc expression following RT. A small minority of older individuals with the most extreme hypertrophic response also increase the number of myonuclei per myofiber following RT, likely to provide more rDNA template for rRNA transcription. Finally, we show that chemical inhibition of de novo Pol I-mediated rRNA transcription completely abolishes growth factor-induced increases in protein synthesis and myotube hypertrophy in vitro. Previously, it was thought that increased translational efficiency was the primary regulator of RT-induced muscle hypertrophy. The data presented in this study suggest that ribosome biogenesis is a novel regulatory mechanism that controls RT-induced human skeletal muscle growth.

Comment: Skeletal muscle exists in a state of sensitive adaptation to expected usage, a consequence of its critical role in survival combined with its comparatively high basal energy requirements. Insufficiently exercised muscle atrophies by default, a phenomenon familiar to retired athletes and serving astronauts alike, whereas muscles under modest use remain stable and those under high demand increase in size to better meet future needs. Although the equilibrium between these processes is maintained for most of the contemporary lifespan, the last aspect in particular begins to fail in the elderly–leading to a pathology known as sarcopenia, where any loss of muscle is difficult or impossible to reverse (although sufficient exercise can sustain preexisting mass ¹⁸ ). Clinically defined sarcopenia affects around 5%–10% of those aged over 65, but closer to 50% of those aged 85 or older; the situation is of course considerably worse if one refuses to draw an arbitrary and possibly misleading ¹⁹ line between pathological wasting and “normal aging,” since muscle mass typically begins to decrease around age 30. Sarcopenia causes a progressive loss of strength, balance, ²⁰ and capacity to perform activities of daily living; consequently it is strongly associated with worsened outcomes from physical trauma, as well as with the general trend toward systemic decline referred to as frailty. This work identifies a novel and strikingly fundamental relationship between the decline in hypertrophic function with aging and a deficiency in the generation of new ribosomes within muscle cells. Given the observation that those elderly individuals who avoid said decline do so through upregulation of c-Myc and the frequent involvement of that gene in carcinogenesis, we are tempted to speculate that the phenomenon partly reflects chronic destruction of the most readily Myc-expressing myofibers by the increasingly dysregulated and cross-reactive immune system of older individuals. Regardless of the exact mechanism, however, this work also suggests a previously unappreciated therapy; supplying an adequate surplus of ribosomal RNA and/or DNA to elderly muscles may suffice to restore youthful hypertrophic potential.